Methods for the synthesis of dicarba bridges in organic compounds

ABSTRACT

The present invention relates to methods for forming dicarba bridges in organic compounds. This involves the use of a pair of complementary metathesisable groups on the organic compound, and subjecting the compound to cross-metathesis under microwave radiation conditions. In an alternative, the compounds contain a turn-inducing group between the pair of cross-metathesisable groups to facilitate the cross-metathesis.

1.1 FIELD OF THE INVENTION

The present application broadly relates to methods for forming dicarbabridges in organic compounds, and compounds such as peptides containingdicarba bridges.

1.2 BACKGROUND TO THE INVENTION

Cystine (—S—S—) bridges are common structural motifs in naturallyoccurring cyclic peptides. In some cases, these disulfide bridges act asreactive functional groups. In many other cases however, the cystinebridge serves only a skeletal, structural role, maintaining secondaryand tertiary structure. Disulfide bonds in peptides and other compoundsare highly reactive under broad-ranging conditions, and therefore usefulpeptides containing disulfide bonds which have a structural role are atrisk of denaturation, resulting in loss of properties. There isaccordingly some interest in developing methods for creating more robustbridges in such compounds—such as dicarba (—C—C—) containing bridges,which are not as reactive, so as to produce compounds having theactivity of, or similar activity to, the disulfide-containingpolypeptides, but with better biostability.

Once a suitably strategy for forming such dicarba bridges isestablished, it is of additional interest to be able to form multipledicarba bridges—selectively. By way of explanation, a peptide possessingfour cysteine residues, and two cystine bridges, has threetopoisomers—the [1,3],[2,4]-isomer (globule), the [1,4],[2,3]-isomer(ribbon) and the [1,2],[3,4]-isomer (bead). It would be useful to beable to selectively form one of these isomers, without any of the othertwo topoisomers. It is also of interest to be able to form one or moredicarba bridges using chemistry that does not destroy any disulfidebridges that are present, so that dicarba-disulfide containing compoundscan additionally be formed. It is of further interest to have a dicarbabridge forming method that can take place despite the presence ofdisulfide, which could otherwise interfere with dicarba bridge-formingreactions.

Once this is achievable, it is of interest to be able to formdicarba-containing analogues of a range of disulfide-containingpeptides, such as conotoxins. It is also of interest to form peptide andnon-peptide compounds containing one or more intramolecular dicarbabridge, and an olefin-handle enabling reaction to other moieties.

2.0 SUMMARY OF THE INVENTION

According to the present invention, there is provided a range of methodsfor forming dicarba bridges, as well as new compounds containing dicarbabridges and a range of new compounds that facilitate the construction ofthese bridges.

According to a first aspect, there is provided a method for thesynthesis of an organic compound with a dicarba bridge, comprising:

-   -   providing a reactable organic compound having a pair of        unblocked complementary metathesisable groups, or two or more        reactable organic compounds having between them a pair of        unblocked complementary, metathesisable groups, and    -   subjecting the reactable organic compound or compounds to        cross-metathesis under microwave radiation conditions to form an        organic compound with an unsaturated dicarba bridge.

As explained in further detail below, cross-metathesis involves theformation of an unsaturated dicarba bridge (inter- or intramolecular,depending on whether there are one or two reactable organic compounds)from two unblocked metathesisable olefinic groups. It has beensurprisingly found that for many situations where the reaction will notproceed under normal conditions, the performance of this reaction undermicrowave radiation conditions overcomes this problem and enables thisreaction to proceed. Another strategy for improving the performance ofthe cross-metathesis which does not rely on microwave is outlined below.The advantages of the use of microwave irradiation applies particularlyto the situation where the method is performed on a single reactableorganic compound having a pair of unblocked complementary metathesisablegroups, for the formation of an intramolecular dicarba bridge. In othercases, microwave radiation overcomes inefficient metathesis reactionsthat do not otherwise go to completion. Other details relating to thetypes of compounds that this method is particularly suited to areoutlined in the detailed description.

In a related aspect, in which it is desired to form a saturated dicarbabridge, the process involves a following step of subjecting theunsaturated dicarba bridge to hydrogenation (suitably homogeneoushydrogenation). Accordingly, in total, this second aspect provides amethod for the synthesis of an organic compound with a saturated dicarbabridge, comprising:

-   -   providing a reactable organic compound having a pair of        unblocked complementary metathesisable groups, or two or more        reactable organic compounds having between them a pair of        unblocked complementary metathesisable groups,    -   subjecting the reactable organic compound or compounds to        cross-metathesis under microwave radiation conditions to form an        organic compound with unsaturated dicarba bridge, and    -   subjecting the unsaturated dicarba bridge to hydrogenation.

The hydrogenation step is suitably homogeneous hydrogenation.

According to one particularly preferred embodiment, the process enablesthe selective formation of multiple dicarba bridges. According to thisembodiment, there is provided a method for the synthesis of an organiccompound with a plurality of dicarba bridges, comprising:

-   -   providing one or more reactable organic compounds having within        the single compound, or between the multiple compounds, a first        pair of complementary metathesisable groups which are unblocked,        a second pair of complementary metathesisable groups, which are        blocked and can be unblocked by an unblocking reaction or series        of reactions specific to that second pair, and optionally        further pairs of complementary methathesisable groups, which are        blocked and can be unblocked by an unblocking reaction or series        of reactions specific to each further pair,    -   subjecting the reactable organic compound or compounds to        cross-metathesis to form an organic compound with an unsaturated        dicarba bridge across the first pair of complementary        metathesisable groups, without cross-metathesis between the pair        or pairs of blocked complementary metathesisable groups,    -   subjecting the second pair of complementary metathesisable        groups to the unblocking reaction or series of reactions        specific to the second pair,    -   subjecting the second pair of complementary metathesisable        groups to cross-metathesis to form an organic compound with an        unsaturated dicarba bridge across the second pair of        complementary metathesisable groups, without cross-methathesis        between any pair or pairs of complementary methathesisable        groups that remain blocked, and    -   if any complementary metathesisable groups remain, subjecting        those groups to unblocking reactions specific to those pairs,        followed by cross metathesis, wherein at least one of the        cross-metathesis reactions is conducted under microwave        radiation conditions.

Preferably, the unblocking reaction specific to the second paircomprises cross-metathesis with a butadiene-free disposable olefin.1,3-butadiene acts as a poison in the unblocking reaction, if it ispresent in the disposable olefin composition used in this reaction.

In many circumstances it will be desirable to subject some or all of theunsaturated dicarba bridges formed by cross-metathesis to hydrogenation.This can be completed in stages following each cross-metathesis, or itmay be conducted as a single hydrogenation step for converting allunsaturated dicarba bridges present at that point into saturated dicarbabridges (following two or more cross-metathesis reactions). Byconvenient selection of the appropriate time at which to perform thehydrogenation(s), it is possible for selected dicarba bridges to besaturated and for other dicarba bridges to remain unsaturated. Thehydrogenation step(s) is/are suitably homogeneous hydrogenation.

Thus, where all dicarba bridges are desired to be saturated, the processdescribed above may comprise the further steps of:

-   -   subjecting the unsaturated dicarba bridge formed between the        first pair of complementary metathesisable groups to        hydrogenation, and    -   subjecting the unsaturated dicarba bridge formed between the        second pair of complementary metathesisable groups to        hydrogenation, wherein each homogenous hydrogenation is        performed either separately or in the one step.

According to one embodiment, the hydrogenation of the complementarymethathesisable groups takes place immediately after cross-metathesis ofthat pair of complementary metathesisable groups. The hydrogenation issuitably a homogeneous hydrogenation.

It is an option to perform each intramolecular cross-metathesis reactionunder microwave radiation conditions.

In the detailed description, a particularly suitable series of reactionsappropriate to the formation of two and three dicarba bridges isdescribed.

The method of the present invention is particularly suited to theformation of peptides with dicarba bridges. In this event, the reactablecompound, or one of the reactable compounds, is attached to a solidsupport. Suitable conditions for performing the reaction, taking intoaccount the difficulties that are introduced as a result of conductingthe reaction on a solid support, are described in the detaileddescription. It is noted however that compounds other than peptides canalso suitably be prepared through a reactable compound which is attachedto a solid support, using the microwave cross-metathesis reactionconditions.

A strategy that is an alternative to microwave irradiation has beendevised for improving the performance of a cross-metathesis between twocomplementary metathesisable groups (olefins) in the one organiccompound.

According to this embodiment, there is provided a method for thesynthesis of an organic compound with a dicarba bridge, comprising:

-   -   synthesising a reactable organic compound to contain a pair of        unblocked complementary metathesisable groups, and a        turn-inducing group in between the pair of complementary        metathesisable groups, and    -   subjecting the reactable organic compound to cross-metathesis to        form a compound with an unsaturated dicarba bridge.

If the target organic compound is to contain a saturated dicarba bridge,the compound is subjected to hydrogenation (suitably homogeneoushydrogenation).

This method is particularly suited to the synthesis of peptides withdicarba bridges.

The present invention also provides for a compound produced by themethod of the invention. The compound may be a peptide with at least onedicarba bridge, or may be any other organic compound with a dicarbabridge.

2.1 BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a reaction scheme demonstrating some possible locations forthe complementary metathesisable groups, in a peptide.

FIG. 2 is a ¹H n.m.r. spectrum for assessing binding between dienamide57 and catalyst, forming a ruthenium-vinylalkylidene complex 73(spectrum a), a new species 74 (spectrum b) after 60 minutes, andcomplex 74 (spectrum c) after 18 hours.

FIG. 3 is a ¹H n.m.r. spectrum of compounds 83, 19 and 82, showingseparation of characteristic peaks for each.

FIG. 4 is a graph of catecholamine release from dicarba-conotoxims 118and 119.

FIG. 5 is a gas chromatogram trace for commercial trans 2-butene,showing trans 2-butene (A), cis 2-butene (B) and catalyst poison1,3-butadiene.

3.0 DETAILED DESCRIPTION

As described above, this application relates to the formation of organiccompounds containing dicarba bridges.

3.1 Types of Compounds and Groups

The term organic compound is used in its broadest sense to refer toorganic, carbon-containing compounds, as opposed to inorganic compoundsthat are not based on carbon. To the extent that the method can be usedto prepare organic ligands for organometallic compounds, this is alsoencompassed. Specific examples of organic compounds that the inventionis particularly suited to are peptides.

The term “peptide” is used in this specification in its broadest senseto refer to oligomers of two or more amino acids. The term “side chain”is used in the usual sense to refer to the side chain on the amino acid,and the backbone to the H₂N—(C)_(x)—CO₂H (where x=1, 2 or 3) component,in which the carbon in bold text bears the side chain (the side chainbeing possibly linked to the amino nitrogen, as in the case of proline).

One class of peptides of interest are the peptidomimetics—that is, apeptide that has a series of amino acids that mimics identically orclosely a naturally occurring peptide, but with at least one dicarbabridge, and optionally one or more further differences, such as theremoval of a cystine bridge, a change by up to 20% of the amino acids inthe sequence, as non-limiting examples. Of particular interest aredicarba analogues of naturally-occurring disulfide-containing peptides,in which one or more of the disulfide bonds are replaced with dicarbabridges. These may also be classed as pseudo-peptides.

The term “amino acid” is used in its broadest sense and refers to L- andD-amino acids including the 20 common amino acids such as alanine,arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine andvaline (illustrated in the Appendix); and the less common amino acidderivatives such as homo-amino acids, N-alkyl amino acids, dehydroaminoacids, aromatic amino acids and α,α-di substituted amino acids, forexample, cystine, 5-hydroxylysine, 4-hydroxyproline, α-aminoadipic acid,α-amino-n-butyric acid, 3,4-dihydroxyphenylalanine, homoserine,α-methylserine, ornithine, pipecolic acid, ortho, meta orpara-aminobenzoic acid, citrulline, canavanine, norleucine, δ-glutamicacid, aminobutyric acid, L-fluorenylalanine, L-3-benzothienylalanine andthyroxine; β-amino acids (as compared with the typical α-amino acids)and any amino acid having a molecular weight less than about 500. Theterm also encompasses amino acids in which the side chain of the aminoacid comprises a metathesisable group, as described herein. Further, theamino acid may be a pseudoproline (ψPro).

The amino acids may be optionally protected. The term “optionallyprotected” is used herein in its broadest sense and refers to anintroduced functionality which renders a particular functional group,such as a hydroxy, amino, carbonyl or carboxyl group, unreactive underselected conditions and which may later be optionally removed to unmaskthe functional group. A protected amino acid is one in which thereactive substituents of the amino acid, or the amino group or carboxylgroup of the amino acid are protected. Suitable protecting groups areknown in the art and include those disclosed in Greene, T. W.,“Protective Groups in Organic Synthesis” John Wiley & Sons, New York1999, (the contents of which are incorporated herein by reference) asare methods for their installation and removal.

Preferably the N-protecting group is a carbamate such as,9-fluorenylmethyl carbamate (Fmoc), 2,2,2-trichloroethyl carbamate(Troc), t-butyl carbamate (Boc), allyl carbamate (Alloc),2-trimethylsilylethyl (Teoc) and benzyl carbamate (Cbz), more preferablyFmoc.

The carboxyl protecting group is preferably an ester such as an alkylester, for example, methyl ester, ethyl ester, t-Bu ester or a benzylester.

The amino acids may be protected, for example, the carboxyl groups ofaspartic acid, glutamic acid and α-aminoadipic acid may be esterified(for example as a C₁-C₆ alkyl ester), the amino groups of lysine,ornithine and 5-hydroxylysine, may be converted to carbamates (forexample as a C(═O)OC₁-C₆ alkyl or C(═O)OCH₂Ph carbamate) or imides suchas thalimide or succinimide, the hydroxyl groups of 5-hydroxylysine,4-hydroxyproline, serine, threonine, tyrosine,3,4-dihydroxyphenylalanine, homoserine, α-methylserine and thyroxine maybe converted to ethers (for example a C₁-C₆ alkyl or a (C₁-C₆alkyl)phenyl ether) or esters (for example a C═OC₁-C₆ alkyl ester) andthe thiol group of cysteine may be converted to thioethers (for examplea C1-C6 alkyl thioether) or thioesters (for example a C(═O)C₁-C₆ alkylthioester).

The term “dicarba bridge” is used broadly, unless the context indicatesotherwise, to refer to a bridging group that includes the sequence—C—C—. This encompasses both the unsaturated (—C═C—) and saturated(—C—C—) dicarba sequence. The atoms directly attached to the carbonatoms of the dicarba sequence (—C—C—) are typically H, although furtheror alternative reactions can be performed to introduce substituentsother than hydrogen onto the carbon atoms of the dicarba sequence of thedicarba bridge. Hydrogenated dicarba bridge refers to the specific casewhere the dicarba bridge is —CH₂—CH₂—. The term unsaturated hydrogendicarba bridge is used to refer to —CH═CH—. This may be cis- or trans-ingeometry.

In addition to the dicarba sequence, the dicarba bridge may include anyother series of atoms, typically selected from C, N, O, and P, althoughthe atoms to either side of the dicarba sequence are preferably carbon,and with the proviso that the nitrogen atoms present in the compoundduring metathesis are not free amines (protected amines, such ascarbamates, are acceptable). Thus, the dicarba bridge encompasses thefollowing possible bridges, as illustrative examples:

In IV, R₁ and R₂ are each independently selected from any divalentlinking group. Such divalent linking groups should not be groups thatpoison the metathesis catalyst. Most free amines poison metathesiscatalysts and therefore are preferably protected or avoided duringmethathesis.

The dicarba bridge may form a bridge between two separate reactableorganic compounds, to form an intermolecular bridge, or it may form abridge between two points in a single reactable organic compound, so asto form an intramolecular bridge, otherwise known as a ring. It isparticularly difficult to form intramolecular bridges, due to sterichindrance, and the need to bring the reactable (metathesisable) groupstogether. The use of microwave radiation in the cross-metathesis stephas enabled this to occur, or occur more efficiently.

“Reactable organic compound” is a term used to refer to the organiccompound that is subjected to the reaction, as distinct from the targetorganic compound, to facilitate understanding of which “organiccompound” is being referred to in the process. The “reactable” organiccompound is therefore any compound that can be subjected to the reactiondescribed, and using other terminology may be considered to be astarting material, an intermediate, a reagent or otherwise.

In this specification, including the claims which follow, except wherethe context requires otherwise due to express language or necessaryimplication, the word “comprising” or variations such as “comprise” or“comprises” is used in the inclusive sense, to specify the presence ofthe stated features or steps but not to preclude the presence oraddition of further features or steps.

As used in the specification, the words “a”, “an” and “the” include theplural equivalents, unless the context clearly indicates otherwise.Thus, for example, reference to “an amino acid” includes one or moreamino acids.

The method for the formation of dicarba bridges involves the use ofcomplementary pairs of metathesisable groups on a compound.

3.2 Cross-Metathesis

Cross-metathesis is a type of metathesis reaction involving theformation of a single olefin bond across two unblocked, or reactiveolefins, to form a new olefinic bridge spanning across the two reactiveolefins. In a general sense, metathesis can be described as the mutualintermolecular exchange of alkylidene (or carbene) fragments between twoolefins promoted by metal-carbene complexes. The cross-metathesis isconducted with a metathesis catalyst. There are many metathesiscatalysts known in the art. Examples of suitable catalysts are theruthenium catalysts, such as Grubbs' catalyst—first and secondgeneration. For details of other suitable cross-metathesis catalysts,reference is made to Grubbs, R.H. Handbook of Metathesis; Wiley-VCH: NewYork, 2003; 1204 pages, 3 volumes, the entirety of which is incorporatedby reference. New catalysts are being developed all the time, and any ofthese new cross-metathesis catalysts can be used. For additionalinformation on this reaction, and appropriate conditions and catalystsfor the performance of the reaction, reference is also made toChatterjee et al, J. Am, Chem, Soc, 2003, 125, 11360-11370, the entiretyof which is incorporated herein by reference.

Ring-closing metathesis is a particular example of cross-metathesiswhere the two reactive olefins are on the one compound, so as to form anintramolecular bridge, or ring.

3.3 Blocking and Activation

For metathesis to occur between two alkylidenes (olefins), thealkylidenes must not be blocked by any steric or electronic blockinggroups. A steric blocking group is any bulky group that stericallyprevents the metathesis from taking place in the presence of across-metathesis catalyst. Examples of steric blocking groups on anolefin are alkyl. Prenylglycine is an example of an amino acidcontaining a dialkyl-blocked olefin side chain (specifically,dimethyl-blocked). Removal of one or both of the blocking groupsunblocks the olefin, and enables the cross-metathesis to take place. Itis noted that the pair of metathesisable groups that remain afterunblocking need not be identical—a mono-substituted olefin (such as amono-methylated olefin) and an unsubstituted olefin (being unsubstitutedat the open olefinic end) can form a suitable pair ofcross-metathesisable groups. The term “complementary” is used toindicate that the pair of unblocked metathesisable groups are notnecessarily identical, but are merely complementary in the sense thatcross-metathesis can take place across the two olefinic groups.

Electronic blocking refers to the presence of a group on the reactableorganic compound or compounds that modifies the electronic nature of theolefin group of the reactable organic compound (which would otherwiseundergo cross-metathesis), so as to prevent that olefin group fromundergoing cross-metathesis. An example of an electronic blocking groupis a conjugated double bond—that is, a double bond located in an α-βrelationship to the olefinic group that would otherwise undergocross-metathesis. The α-β-unsaturation withdraws electrons away from theolefinic cross-metathesisable group, to cause electronic blockingpreventing cross-metathesis from taking place.

By using a combination of blocking mechanisms, a series of pairs ofcross-metathesisable olefinic groups in the reactable organic compoundor compounds can be designed, with different reaction conditions toeffect selective unblocking of particular pairs. In this way, it becomespossible to regioselectively synthesise multiple dicarba bridges (interand/or intramolecular) in compounds.

3.4.1 Microwave Reaction Conditions

It has been found that when the cross-metathesis reaction is performedunder microwave reaction conditions, the reaction may take place insituations where the reaction would not otherwise take place—forinstance, when the metathesisable groups are unblocked, but thearrangement, length or spatial orientation of the reactable organiccompound prevents the metathesisable groups from being close enough toone another to enable the reaction to proceed. An alternative strategyis described in Section 3.4.2.

The microwave reaction conditions involve applying microwave radiationto the reactable organic compounds in the presence of thecross-metathesis catalyst for at least part of the reaction, usually forthe duration of the reaction. The microwave or microwave reactor may beof any type known in the art, operated at any suitable frequency.Typical frequencies in commercially available microwave reactors are2.45 GHz, at a power of up to 500 W, usually of up to 300 W. Thetemperature of the reaction is preferably at elevated temperature, as aconsequence of the microwave radiation, preferably at reflux, or around100° C., as is appropriate in the case. The reaction is preferablyperformed in a period of not more than 5 hours, suitably for up to about2 hours.

3.4.2 Turn-Inducing Groups

There is a strategy that is an alternative to microwave irradiation thathas been devised for improving the performance of a cross-metathesisbetween two complementary metathesisable groups (olefins) in the oneorganic compound.

According to this embodiment, there is provided a method for thesynthesis of an organic compound with a dicarba bridge, comprising:

-   -   synthesising a reactable organic compound to contain a pair of        unblocked complementary metathesisable groups, and a        turn-inducing group in between the pair of complementary        metathesisable groups, and    -   subjecting the reactable organic compound to cross-metathesis to        form a compound with an unsaturated dicarba bridge.

If the target organic compound is to contain a saturated dicarba bridge,the compound is subjected to hydrogenation (suitably homogeneoushydrogenation).

This method is particularly suited to the synthesis of peptides withdicarba bridges.

Peptides are generally quite linear, as the component amino acids(especially when these are the 20 common amino acids, with exception ofproline) and the backbone of the peptide, is linear. Proline, with thering structure linking to the amino nitrogen atom, induces a turn or abend in an otherwise linear peptide. This is a naturally-occurringturn-inducing group. This embodiment is particularly suited to thosepeptides that do not contain a naturally-occurring turn-inducing aminoacid. In this case, a synthetic (non-naturally occurring) turn-inducinggroup is located in the compound—or in the amino acid sequence.

Preferably the turn-inducing group is a turn-inducting amino acid orprotein, and is preferably synthetic (non-naturally occurring). Examplesof suitable synthetic turn-inducing amino acids are the pseudoprolines,including derivatives of serine, threonine and cysteine which have beenderivatised to contain a cyclic group between the amino acid sidechain(via the —OH or —SH group), and the amino nitrogen atom. A typicalderivatising agent is CH₃—C(═O)—CH₃, such that the turn-inducing aminoacids are:

After cross-metathesis, the pseudoproline(s) are converted back to theunderivatised amino acid (serine, threonine or cysteine) by removal ofthe derivatiseing agent. The conditions for cleavage from a solidsupport will achieve this.

According to the present invention, there is provided a method for thesynthesis of a peptide with an intramolecular dicarba bridge, the methodcomprising:

-   -   synthesising a peptide comprising a series of amino acids        supported on a solid support, wherein two amino acids comprise a        first pair of complementary metathesisable groups, and one amino        acid between said amino acids comprising the first pair of        complementary metathesisable groups in the series which is a        turn-inducing amino acid, and    -   subjecting the peptide to cross-metathesis to form a peptide        with an unsaturated dicarba bridge between the amino acids        bearing the metathesisable groups.

The method may further comprise one or more of the following additionalsteps:

-   -   subjecting the unsaturated discarba bridge to hydrogenation to        form a peptide with a saturated intramolecular dicarba bridge;    -   cleaving the peptide from the solid support.

If the turn-inducing amino acid is one of pseudo-serine, pseudo-prolineor pseudo-cysteine, then the method may further comprise the step ofconverting the pseudo-serine, pseudo-proline or pseudo-cysteine toserine, proline or cysteine, respectively.

The process can be combined with the other preferred features describedherein.

3.5 Solvents

Particularly for reactions conducted with the (or one of the) reactableorganic compound(s) attached to a solid support such as a resin, thecross-metathesis is preferably performed in a solvent combination of aresin-swelling solvent, with a co-ordinating solvent for the catalyst.In resin-supported reactions, swelling of the resin is required to avoid“clumping”, but such solvents are not generally compatible withcross-metathesis catalysts. For example, polystyrene-based resins showoptimal swelling in chlorinated solvents such as dichloromethane,however these solvents are not compatible with hydrogenation catalysts.The solvents react with such catalysts to compromise catalystfunction—which in turn reduces the catalytic cycle (or turn-overnumber—TON), resulting in incomplete conversion. It was found that theaddition of a small amount of a co-ordinating solvent for the catalyst,such as an alcohol (methanol, isopropanol, etc) which can co-ordinateinto a vacant site of the catalyst to facilitate stability, overcamethis problem. The co-ordinating solvent is suitably used in an amount ofabout 1-30%, for example constituting 10% of the solvent, by volume. Theresin swelling agent may be any polar solvent known to swell the resin,such as dichloromethane. Other suitable solvents for a range of resinsare as set out in Santini, R., Griffith, M. C. and Qi, M., Tet. Lett.,1998, 39, 8951-8954, the entirety of which is incorporated herein byreference.

3.6 Solid Supports

The (or one of the) reactable organic compound(s) is preferably attachedto a solid support—especially in the case of peptide reactable organiccompound(s). A plethora of solid supports are known and available in theart, and include pins, crowns, lanterns and resins. Examples arepolystyrene-based resins (sometimes referred to as solid supports),including cross-linked polystyrene containing some divinylbenzene (eg1%), functionalised with linkers (or handles) to provide a reversiblelinkage between the reactable organic compound (which may be a peptidesequence containing side-chains with cross-metathesisable groups) andthe resin. Examples are the Wang resin, Rink amide resin,BHA-Gly-Gly-HMBA resin and 2-chlorotrityl chloride resin, which are allpolystyrene-based. Other forms of solid supports that may notnecessarily be characterised as resins can also be used.

It has been surprisingly found that using the microwave reactionconditions, it is possible to have a higher solid support loading thanis conventionally used in peptide synthesis on solid supports. Typicalsolid support loadings are at the 0.1 mmol/g level, but microwaveradiation (optionally combined with solvent choice, as described above)overcomes the aggregation problems at higher solid support loadings, sothat solid support loading at around 0.9 mmol/g (nine times higher) isachievable. As a consequence, one embodiment of the invention relates tothe performance of the reaction at high solid support loadings—that is,at loadings of 0.2 mmol/g and above, such as 0.5 mmol/g and above.

3.7 Hydrogenation

The product of the cross-metathesis reaction is a compound with anunsaturated dicarba bridge. If the target organic compound is to containa saturated dicarba bridge, the process further comprises the step ofsubjecting the unsaturated dicarba bridge to hydrogenation (suitablyhomogeneous hydrogenation).

Hydrogenation of the dicarba bridge is performed with a catalyst that ischemoselective for unblocked non-conjugated carbon-carbon double bonds,as distinct from other double bonds (such as carbon-oxygen double bondsin carbonyl groups and carboxylic acids, and blocked conjugated doublebonds). One notable example of a suitable catalyst is Wilkinson'scatalyst. Wilkinson's catalyst and catalysts like it are not asymmetrichydrogenation catalysts but however as this type of hydrogenation doesnot form a new chiral centre this is acceptable for this form ofhydrogenation reaction. Although the use of asymmetric hydrogenationcatalyst is not necessary in the hydrogenation of the dicarba bridge,asymmetric hydrogenation catalysts can nevertheless be used. Suitablecatalysts are well known in the art, and include the range of catalystsdescribed for this purpose in Ojima, I. Catalytic Asymmetric Synthesis;Wiley-VCH: New York, 2000; Second Edition, Chapter 1, 1-110, theentirety of which is incorporated by reference. New catalysts havingsuch properties are developed from time to time, and these may also beused. Further examples of suitable asymmetric hydrogenation catalystsare the chiral phosphine catalysts, including chiral phospholane Rh(I)catalysts. Catalysts in this class are described in U.S. Pat. No.5,856,525. Such homogenous hydrogenation catalysts are tolerant ofsulfide, and disulfide bonds, so that the presence of disulfide bondsand the like will not interfere with the synthetic strategy. Thehydrogenation can be conducted at any temperature, such as roomtemperature or at elevated temperature. The reaction is typicallyconduced at elevated pressure, although if slower reaction times can betolerated, the reaction can be performed at atmospheric pressure.

In other stages of the process in which hydrogenation is used as astrategy for unblocking complimentary methasisable groups, it may bebeneficial for the hydrogenation catalyst used in those reactions to beasymmetric to stereoselectively form a new chiral centre. Nevertheless,if a racemic mixture can be tolerated, a catalyst such as Wilkinson'scatalyst could be used.

Homogeneous hydrogenation is used in its broadest sense to refer tocatalytic hydrogenations conducted in one phase such as a liquid phase,where the liquid phase contains the substrate molecule/s and solvent.More than one solvent, such as organic/aqueous solvent combinations, orfluorous solvent combinations, non-aqueous ionic pairs, supercriticalfluids, or systems with soluble polymers may also be employed. This isdistinct from heterogeneous reactions, which involve more than onephase—as in the case of hydrogenations performed with solid-supportedcatalysts in a liquid reaction medium.

3.8 Regioselective Formation of Multiple Dicarba Bridges

The strategy for the formation of a dicarba bridge described above canbe combined with other reaction steps for the formation of an organiccompound with a dicarba bridge and a disulfide bridge, or for theformation of organic compounds with multiple dicarba bridges, optionallywith disulfide bridges.

To form a plurality of (i.e. two or more) dicarba bridges, it isnecessary to include at appropriate locations in the reactive organiccompound or compounds pairs of complementary metathesisable groups whichare blocked or deactivated for the times when different pairs ofmetathesisable groups are being linked together, and unblocked or“activated” to enable reaction to occur between those pairs.Accordingly, for each bridge-forming pair, there needs to be anunblocking reaction available that will selectively unblock the requiredpairs.

The first pair to be subjected to the cross metathesis and hydrogenationto form a saturated dicarba bridge need not be blocked during synthesisof the reactive organic compound or compounds. The compound with thispair of unblocked complementary metathesisable groups is then subjectedto the reactions described above to form a dicarba bridge (saturated orunsaturated). Suitable groups for forming the first pair ofcomplementary methathesisable groups which are not blocked are —CH═CH₂—containing organic moieties, and —CH═CH—CH₃— containing moieties. In thecase of peptide synthesis, this may be provided by any amino acidcontaining the side chain —CH═CH₂, optionally with any divalent linkinggroup linking the carbon at the “open” end (the —CH═ carbon atom) to theamino acid backbone, such as an -alkylene-, -alkylene-carbonyl-, and soforth. Examples of —CH═CH₂— containing amino acids and —CH═CH—CH₃—containing amino acids are allyl glycine and crotyl glycine,respectively. Each of these amino acids contains the divalent linkinggroup —CH₂— between the alkylene and the amino acid (peptide) backbone.

At the completion of that reaction, (and optionally after hydrogenationof the first dicarba bridge) the blocked second pair of complementarymetathesisable groups, can be subjected to an unblocking reaction. Thisunblocking reaction involves cross-metathesis with a disposableolefin—which replaces the steric blocking groups on the olefin(metathesisable group) with ═CHR₅, described further below.

Suitable functional groups for forming the second pair of complementarymetathesisable groups are di-blocked alkylenes, such as the group—CH═CR₃R₄, in which R₃ and R₄ are each independently selected from anyblocking groups, such as alkyl, for example methyl. Again, there may bea divalent linking group between the —CH═ carbon atom, and the aminoacid backbone, such as an alkylene group, for instance —CH₂—. An exampleof an amino acid containing this group is prenyl glycine, or protectedprenyl glycine.

The unblocking reaction, or activation reaction, to convert the pair ofdi-blocked alkylenes into an unblocked alkylenes involves subjecting theblocked second pair of complementary metathesisable groups tocross-metathesis with a disposable olefin, to effect removal of theblocking groups (such as R₃ and R₄ in the example shown above).

It will be understood that in this case, cross metathesis is used toreplace the portion ═CR₃H₄ with another unblocked portion ═CH₂ or ═CHR₅,(in which R₅ may be —H, functionalised alkyl or alkyl for instance)which is then “activated” or “unblocked” and ready for being subjectedto cross-metathesis for the formation of a dicarba bridge, using thesame techniques described above.

The conditions for this activation-type of cross-metathesis are the sameas described above for the dicarba bridge forming metathesis. It can beperformed under microwave conditions, although it need not be, as thedisposable olefin is a smaller molecule and less subject to the spatialconstraints as larger reactable organic compounds and single reactableorganic compounds in which intramolecular bridges are to be formed.

The “disposable olefin” is suitably a mono-substituted ethylene (such asmonoalkylated ethylene—such as propene, which is a mono-methylatedethylene), or a 1,2-disubstituted ethylene such as high purity 2-butene(cis, trans or a mixture). Previously, commercial 2-butene has beenattempted to be used as the disposable olefin in this unblockingreaction, and the reaction is thus sometimes referred to as“butenolysis”. However, until now commercially available 2-butene (whichis a mixture of cis- and trans-2-butene) has inexplicably not enabledthe reaction to proceed. As detailed further below, a method has beenfound for overcoming this problematic reaction.

The substituents of the substituted ethylene disposable olefin aresubstituents that do not participate in the reaction. Examples are alkylor a functionalised (substituted) alkyl. The functional group of thefunctionalised alkyl is suitably a polar functional group, to assistwith swelling of the solid support, and solubility. Examples arehydroxy, alkoxy, halo, nitrile and carboxylic acids/esters. One specificexample is the di-ester functionalised disposable olefin1,4-diacetoxy-2-butene.

Thus the disposable olefin is suitable a 1,3-butadiene-free disposableolefin, or a 1,3-butadiene-free mixture of disposable olefin and ispreferably 1,3-butadiene-free olefin or olefin mixture of one or more ofthe following olefins:

wherein X and Y are each independently selected from the groupconsisting of —H, alkyl and alkyl substituted with one or moresubstituents selected from halo, hydroxy, alkoxy, nitrile, acid andester.

Preferably, at least one of X and Y is not H.

Preferably, in the case of the alkyl substituents, the substituent ispreferably on the carbon atom. Preferably the substituted alkyl is asubstituted methyl. According to one embodiment, at least one of X and Yis a substituted alkyl, such as a substituted methyl. X and Y may be thesame or different. The olefins may be cis or trans, or mixtures of both.

3.9 Peptide Synthesis

For the synthesis of a peptide with an intramolecular dicarba bridge,the method may comprise:

-   -   providing a peptide comprising a series of amino acids supported        on a resin, wherein two amino acids comprise sidechains with a        first pair of complementary metathesisable groups which may be        blocked or unblocked;    -   unblocking the first pair of complementary metathesisable        groups, if said groups are blocked; and    -   subjecting the peptide to cross-metathesis under microwave        radiation conditions to form a peptide with an unsaturated        dicarba bridge between the amino acids bearing the        metathesisable groups.

The method may further comprise the step of:

-   -   subjecting the unsaturated dicarba bridge to hydrogenation        (suitably homogeneous hydrogenation), to form a peptide with a        saturated intramolecular dicarba bridge.

Generally, the peptide will be a protected peptide (such as Fmocprotected). The amino acids can be any of the amino acids describedearlier, but it is convenient for the synthesis of peptidomimetics forthe amino acids to be selected from the 20 naturally-occurring aminoacids, γ- and β-amino acids and from any cross-metathesisablegroup-bearing analogues thereof. An example of a cross-metathesisablegroup-bearing analogue is allyl glycine.

The peptide may also be formed so as to have a disulfide bridge inaddition to one or more dicarba bridges. According to this embodiment,the reactable peptide comprises two protected cysteine residues, and themethod comprises deprotecting the cysteine residues and oxidising thecysteine residues to form a disulfide bridge. This may be performed atany stage, such as before the formation of the dicarba bridge(s) orafter. This step can be combined with the processes described in thefollowing for the formation of two or three dicarba bridges and adisulfide bridge. It is noted that the cysteine residues may be locatedon the first peptide, on the second peptide (when present) or on a thirdpeptide.

For the synthesis of a peptide with two intramolecular bridges, themethod comprises:

-   -   providing a first peptide comprising a series of amino acids        supported on a resin, wherein two amino acids comprise        sidechains with a first pair of complementary metathesisable        groups and two amino acids comprise sidechains with a second        pair of blocked complementary metathesisable groups,    -   subjecting the peptide to cross-metathesis under microwave        radiation conditions to form a peptide with an unsaturated        dicarba bridge between the amino acids that bore the first pair        of complementary metathesisable groups,    -   unblocking the second pair of complementary metathesisable        groups, and    -   subjecting the peptide to cross-metathesis to form a peptide        with an unsaturated dicarba bridge between the amino acids that        bore the second pair of complementary metathesisable groups.

As described previously, one or both unsaturated dicarba bridges formedbetween the amino acids that bore the first and second pairs ofcomplementary metathesisable groups may be subjected to homogenoushydrogenation, separately or at the same time.

For the synthesis of a peptide with the intramolecular bridge, and asecond bridge which is an intermolecular, the method comprises:

-   -   providing a first peptide comprising a series of amino acids        supported on a resin, wherein two amino acids comprise        sidechains with a first pair of complementary metathesisable        groups which may be blocked or unblocked, and one amino acid        comprises a sidechain with a second metathesisable group which        may be blocked or unblocked, with the proviso that the        metathesisable groups out of at least one of the first or the        second metathesisable groups are blocked;

-   (a) unblocking the first pair of complementary metathesisable    groups, if said groups are blocked;    -   subjecting the peptide to cross-metathesis under microwave        radiation conditions to form a peptide with an unsaturated        dicarba bridge between the amino acids bearing the first pair of        complementary metathesisable groups, to form a peptide with an        intramolecular dicarba bridge, and

-   (b) contacting the first peptide with a second peptide comprising    one amino acid with a metathesisable group complementary to the    second metathesisable group on the first peptide;    -   unblocking the second complementary metathesisable groups, if        the second metathesisable groups are blocked;    -   subjecting the peptide to cross-metathesis to form a peptide        with an unsaturated dicarba bridge between the amino acids        bearing the second pair of complementary metathesisable groups,        to form a dicarba bridge between the amino acids that bore the        second metathesisable groups,        wherein steps (a) and (b) are performed in either order, so as        to form a peptide with an intermolecular bridge and an        intramolecular bridge.

The method may further comprise the step or steps of subjecting one orboth of the products of step (a) and step (b) to hydrogenation (suitablyhomogeneous hydrogenation) to form a peptide with a saturatedintramolecular dicarba bridge and/or a saturated intermolecular dicarbabridge.

These methods may be combined with a third stage of bridge-formation, toform a peptide with three bridges, one two or three of which areintramolecular. This is achieved by providing a third pair ofmetathesisable groups in the first peptide, or one in the first peptideand one in the second or in a third peptide to be coupled to the firstpeptide through an intermolecular bridge, and then subjecting the thirdpair of metathesisable groups to unblocking to form the compound. Inanother alternative, a complimentary metathesisable group can be “added”to the first or second peptide through the addition of an amino acid orpeptide fragment bearing the metathesisable group. This is illustratedin FIG. 1.

For the formation of a peptide with three intramolecular bridges, themethod comprises:

-   -   providing a first peptide comprising a series of amino acids        supported on a resin, wherein two amino acids comprise        sidechains with a first pair of complementary metathesisable        groups, two amino acids comprise sidechains with a second pair        of blocked complementary metathesisable groups and two amino        acids comprise sidechains with a third pair of blocked        complementary metathesisable groups,    -   subjecting the peptide to cross-metathesis under microwave        radiation conditions to form a peptide with an unsaturated        dicarba bridge between the amino acids bearing the first pair of        complementary metathesisable groups,    -   optionally subjecting the unsaturated dicarba bridge to        hydrogenation,    -   unblocking the second pair of complementary metathesisable        groups,    -   subjecting the peptide to cross-metathesis to form a peptide        with an unsaturated dicarba bridge between the amino acids that        bore the second pair of complementary metathesisable groups,    -   optionally subjecting the unsaturated dicarba bridge to        hydrogenation,    -   unblocking the third pair of complementary metathesisable        groups,    -   subjecting the peptide to cross-metathesis to form a peptide        with an unsaturated dicarba bridge between the amino acids that        bore the third pair of complementary metathesisable groups, and    -   optionally subjecting the unsaturated dicarba bridge to        hydrogenation.

Each of these techniques for the synthesis of peptides with one or moreintramolecular bridges may be combined with additional steps for theformation of one or more intramolecular disulfide bridges.

In each of these techniques it is also preferred that the unblockingreaction specific to the second pair of complementary metathesisablegroups comprise cross-metathesis with a 1,3-butadiene free disposableolefin.

It will be appreciated that if a peptide sequence is added later throughan intermolecular bridge, the corresponding metathesisable groups onthat peptide need not be blocked—as they can be added to the reaction atthe time of cross-metathesis, after the unblocking of the groups on theresin-supported peptide.

3.10 Products of Methods

The present invention also provides for a compound produced by themethod of the invention. The compound may be a peptide with at least onedicarba bridge, or may be any other organic compound with a dicarbabridge. Salts, solvates, derivatives, isomers and tautomers areencompassed in this context.

Possible products include the dicarba analogues of cystine-containingpeptides. Dicarba analogues refers to peptides contain the same aminoacid sequence as the native peptide, but with one or more of the bridgedcysteine-amino acid residue pairs substituted with amino acids bearing adicarba bridge. “Native” is a term used to refer to the natural orsynthetic analogue of a natural peptide—to be distinguished from thedicarba analogue being synthesised. Bis- and higher dicarba analoguesare of particular interest, in view of the difficulty in synthesisingsuch compounds. Examples are the dicarba analogues of Conotoxin ImIpresented in FIG. 6.4. These include the fully dicarba-substitutedanalogues (the final three compounds in that figure) and the partialdicarba analogues (identified as “hybrids” in FIG. 6.4). Other suitableterminology is the mono-dicarba analogues (in which one disulfide bridgeis replaced with a dicarba bridge), and the bis-dicarba analogues (tworeplaced). Thus, the present application also relates to dicarbaanalogues of Conotoxin, including the bis-dicarba, cystino-dicarba andhigher-dicarba analogues.

In FIG. 6.4, the residue between the bridges is represented as“Hag”—based on its synthesis via this amino acid, although the doublebond of Hag is no longer present. In some cases the new bridge isunsaturated and bears a new double bond; in other cases the bridge issaturated. If the peptide was synthesised via another amino acid, suchas crotyl glycine (Crt), Crt would appear in place of Hag. In fact, thepeptides are identical irrespective of whether they were synthesised viaone of these amino acids or the other, as the dicarba bridge is all thatremains from those starting amino acids. Accordingly, the amino acidindicated in the formula for the peptide should not be read as limitingthe peptide to one made specifically through that amino acid. Sub(representing the amino acid suberic acid, which has the cyclised sidechain —(CH₂)₄—) could also have been used to represent the same peptide.

“Conotoxin” is used in its broadest sense to refer to the peptides orpeptide fragments that are present in the venom of cone snails of thegenus Conus (Conidae). All species which are encompassed within thisgenus [class] are contemplated, including the species Conus imperialis,Conus geographus, Conus textile, Conus amadis, Conus tulipa, Conusmarmoreus, Conus lynceus, Conus armadillo, Conus geographus and soforth. The peptides within this class include natural and syntheticpeptides, and derivatives of the naturally-occurring peptides andpeptide fragments. Conotoxins are classified according to their receptorsubtype specificity and the arrangement of disulfide bonds and resultantloop sizes. The paralytic components of the venom (the conotoxins) thathave been the focus of recent investigation are the α-, ω- andμ-conotoxins. All of these conotoxins act by preventing neuronalcommunication, but each targets a different aspect of the process toachieve this. The α-conotoxins target nicotinic ligand gated channels,and the μ-conotoxins target the voltage-gated sodium channels and theω-conotoxins target the voltage-gated calcium channels. Of particularinterest here are the α-, χ- and ω-conotoxins, which contain two orthree disulfide bridges, although μ-conotoxins, δ-conotoxins,κ-conotoxins π-conotoxins and conatokins are also relevant. Theconotoxins are generally between 12 and 30 amino acid residues inlength.

The salts of compounds are preferably pharmaceutically acceptable, butit will be appreciated that non-pharmaceutically acceptable salts alsofall within the scope of the present invention, since these are usefulas intermediates in the preparation of pharmaceutically acceptablesalts. Examples of pharmaceutically acceptable salts include salts ofpharmaceutically acceptable cations such as sodium, potassium, lithium,calcium, magnesium, ammonium and alkylammonium; acid addition salts ofpharmaceutically acceptable inorganic acids such as hydrochloric,orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric,sulfamic and hydrobromic acids; or salts of pharmaceutically acceptableorganic acids such as acetic, propionic, butyric, tartaric, maleic,hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic,succinic, oxalic, phenylacetic, methanesulphonic,trihalomethanesulphonic, toluenesulphonic, benzenesulphonic, salicylic,sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic,lauric, pantothenic, tannic, ascorbic and valeric acids.

In addition, some of the compounds may form solvates with water orcommon organic solvents. Such solvates are encompassed within the scopeof the invention.

By “derivative” is meant any salt, hydrate, protected form, ester,amide, active metabolite, analogue, residue or any other compound whichis not biologically or otherwise undesirable and induces the desiredpharmacological and/or physiological effect. Preferably the derivativeis pharmaceutically acceptable.

The term “tautomer” is used in its broadest sense to include compoundswhich are capable of existing in a state of equilibrium between twoisomeric forms. Such compounds may differ in the bond connecting twoatoms or groups and the position of these atoms or groups in thecompound.

The term “isomer” is used in its broadest sense and includes structural,geometric and stereo isomers. As the compounds that may be synthesisedby these techniques may have one or more chiral centres, it is capableof existing in enantiomeric forms.

3.11 New Reagents to Facilitate Production of Dicarba Bridge ContainingPeptides

The present applicant has synthesised amino acids that are particularlyuseful as they enable the formation of a dicarba bridge when included ina peptide sequence. These amino acids include prenyl glycine in whichthe amino group is protected with a base-removable carbamate-protectinggroup. A particular example of this compound is Fmoc-protected prenylglycine. Fmoc-protected prenyl glycine is a protected, blockedolefin-containing amino acid, suitable for forming the “second” of thedicarba bridges in a peptide, and its synthesis is achieved through theuse of a specific reagent.

Fmoc-protected prenyl glycine requires preparation through thecross-metathesis of Fmoc-protected allyl glycine with 2-alkyl-2-butylene(such as 2-methyl-2-butylene) in the presence of a cross-metathesiscatalyst. The reaction is not complete when isobutylene is used as theolefin in the reaction. The reaction is suitably conducted at a pressureabove 5 psi—preferably at 8 psi or above—for instance at about 10 psi.

4.0 CONTROLLED SYNTHESIS OF (S,S)-2,7-DIAMINOSUBERIC ACID A Method forthe Regioselective Construction of Dicarba Analogues ofDicystine-Containing Peptides

This section describes a solution phase model study for the developmentof a methodology that enables the regioselective formation of dicarbaisosteres of cystine bonds. We investigated a sequence ofruthenium-catalysed metathesis and rhodium-catalysed hydrogenationreactions of non-proteinaceous allylglycine derivatives to achieve highyielding and unambiguous formation of two dicarba bridges. This theorycan also be applied to the synthesis of non-peptide compounds with 2 or3 dicarba bridges.

4.1 Initial Strategy

Our initial strategy planned to capitalise on the use ofα-N-acyl-dienamide 57, a masked precursor to allylglycinederivatives.^(118,119) We devised a strategy involving a doublemetathesis-hydrogenation sequence (Scheme 4.1). This required aselective ring closing metathesis of allylglycine units in the presenceof dienamide functionalities. Grubbs et al. have previously reportedthat selective cross metathesis can be accomplished with olefins ofvarying reactivity.^(130,182) Terminal olefins such as allylglycineundergo rapid homodimerisation with both Grubbs' catalyst¹²⁰ and secondgeneration Grubbs' catalyst,¹²¹ whereas the electron-deficientα-N-acyl-dienamide 57 should be considerably less reactive. Subsequentasymmetric hydrogenation of the dienamide moieties would lead toreactive allylglycine units which could undergo ring closing metathesisto produce the second carbocycle. The final step in this catalyticsequence involves hydrogenation of the unsaturated carbocycles, ifrequired, to afford the saturated cystine isosteres.

In order to validate the proposed strategy, we needed to show that: i)the dienamide 57 would not react under conditions required for the ringclosing metathesis of allylglycine residues, ii) asymmetrichydrogenation of the dienamide 57 would proceed in a highlyregioselective and stereoselective manner, iii) ring closing metathesisof the resulting allylglycine units would proceed in the presence of anunsaturated carbocycle (without resulting in mixed cross metathesisproducts), and iv) the unsaturated carbocycles could be reduced toafford saturated dicarba bridges. We therefore conducted a series ofindependent experiments that would serve as a model to the peptidesystem.

4.1.1 Synthesis of Olefinic Moieties

The dienamide 57 was synthesised according to a literature procedurereported by Teoh et al.¹¹⁹ from a Homer-Emmons olefination of aphosphonate ester 39 and an α,β-unsaturated aldehyde 58 (Scheme 4.2).

The phosphonate, methyl 2-N-acetylamino-2-(dimethoxyphosphinyl)acetate39, was prepared in three steps from commercially available acetamide 34and glyoxylic acid 41.

A mixture of commercially available acetamide 34 and glyoxylic acid 41was heated at reflux in acetone to give pure N-acetyl-2-hydroxyglycine42 as a viscous yellow oil in quantitative yield. The ¹H n.m.r. spectrumsupported formation of the α-hydroxyglycine derivative 42 with theappearance of a methine (H2) doublet and broad amide (NH) doublet at δ5.39 and δ 8.65 respectively. Spectroscopic data were in agreement withthose reported in the literature.¹⁹⁵

Treatment of N-acetyl-2-hydroxyglycine 42 with a catalytic amount ofconcentrated sulfuric acid in methanol furnished methylN-acetyl-2-hydroxyglycinate 43 in 60% yield. These reaction conditionsconverted the carboxylic acid to the methyl ester and the hydroxylfunctional group to methyl ether.

Modification of the reported work-up procedure led to a significantlyimproved yield to that reported in the literature (32%).¹⁹⁶ The presenceof two new methoxyl peaks in the ¹³C n.m.r. spectrum at δ 53.0 and δ56.8 and the corresponding methyl singlets in the ¹H n.m.r. spectrum atδ 3.47 and δ 3.82 supported formation of the desired product 43.Spectroscopic data were also in agreement with those reported in theliterature.¹⁹⁶

The final step in the synthesis of methyl2-N-acetylamino-2-(dimethoxyphosphinyl)-acetate 39 involved reaction ofmethyl N-acetyl-2-hydroxyglycinate 43 with phosphorous trichloride toproduce the intermediate α-chloro ester. Nucleophilic attack of thenewly introduced chlorine substituent with trimethyl phosphite gavephosphonate ester 39 as a colourless solid in low yield (14%). The highsolubility of the ester in water initially led to poor mass recovery.The use of continuous extraction partially overcame this problem and ledto isolation of the product in satisfactory yield (44%).

The ¹H n.m.r. spectrum confirmed formation of the target compound 39with the appearance of a doublet of doublets attributed to the methine(H2) proton coupling to the phosphorous (J=22.2 Hz) and amide proton(J=8.8 Hz). The ¹³C n.m.r. spectrum displayed similar behaviour with themethine (C2) peak appearing as a doublet with large coupling to thevicinal phosphorous atom (J=146.8 Hz). The melting point of the isolatedsolid (89-91° C.) was consistent with that reported in the literature(88-89° C.).¹⁹⁷

(2Z)-Methyl 2-N-acetylaminopenta-2,4-dienoate 57 was synthesised byHomer-Emmons olefination of methyl2-N-acetylamino-2-(dimethoxyphosphinyl)acetate 39 with commerciallyavailable acrolein 58 in the presence of tetramethylguanidine (TMG)(Scheme 4.4). Hydroquinone was added to prevent polymerisation ofacrolein 58 and was found to be critical to the success of thisreaction. The reaction requires the addition of base to a solution ofphosphonate 39 in tetrahydrofuran to generate the carbanion 45, whichwas then reacted with aldehyde 58 to afford the dienoate 57 as anoff-white solid in 85% yield (Scheme 4.4).

The ¹H n.m.r. spectrum of the product supported formation of thedienamide 57 with the appearance of signals corresponding to a newterminal olefin. Doublets at δ 5.49 and δ 5.61 for H5-E and H5-Zrespectively, and an olefinic methine (H4) multiplet at δ 6.47 wereconsistent with formation of dienamide 57. The melting point of theisolated solid (60-62° C.) was also in agreement with that reported inthe literature (61-63° C.).¹¹⁹

Our group have demonstrated that high regioselectivity andenantioselectivity can be achieved in the asymmetric hydrogenation ofdienamide esters. In this case, hydrogenation of dienamide 57 waseffected with Rh(I)—(S,S)-Et-DuPHOS under 30 psi of hydrogen in benzenefor 3 hours (Scheme 4.5). Over-reduction of the terminal olefinic bondwas minimal (<3% 59) under these mild conditions. The (S)-configurationwas determined based on literature assignment for the sametransformation¹¹⁸ and a comparative optical rotation sign to thatreported in the literature for (2S)-methyl 2-N-acetylaminopent-4-enoate21a.²⁰⁸

Asymmetric hydrogenation of dienamide 57 was also performed withRh(I)—(R,R)-Et-DuPHOS to facilitate enantiomeric excess assessment. Thereaction proceeded in quantitative conversion and <5% over-reducedproduct was detected. Chiral GC indicated the reactions proceeded withexcellent enantioselectivity (95% e.e.).

¹H n.m.r. spectroscopy showed the replacement of an olefinic methine(H3) doublet at δ 7.05 with a methylene (H3) multiplet at δ 2.43-2.62.The ¹³C n.m.r. spectrum also displayed new methine (C2) and methylene(C3) peaks at δ 51.8 and δ 36.5 respectively. Spectroscopic data were inagreement with those reported in the literature.¹¹⁹

4.1.2 Cross Metathesis: Homodimerisation

Homodimerisation is a type of cross metathesis in which an olefinself-couples. Conveniently, the only byproduct is a low molecular weightvolatile olefin which is most commonly ethylene (Scheme 4.6).

The mechanism involves an intermolecular exchange of alkylidenefragments between the metal-carbene catalyst and the reacting olefin. Anunstable metallocyclobutane intermediate then decomposes to release thehomodimer and a volatile olefinic byproduct (Scheme 4.7).

Quantitative homodimerisation of allylglycine derivative 21a wasachieved using Grubbs' catalyst in dichloromethane heated at reflux(Scheme 4.8). Purification of the crude product by flash chromatographygave the target compound, (2S,7S)-dimethyl2,7-N,N′-diacetylaminooct-4-enedioate 60, as a brown oil in 88% yield.

High resolution mass spectrometry confirmed formation of the desiredproduct 60 with the appearance of a molecular ion plus sodium peak atm/z 337.1375 for the expected molecular formula (C₁₄H₂₂N₂NaO₆). Inaddition, the ¹³C n.m.r. spectrum displayed a new olefinic methine (C4,5) peak at δ 128.8, whilst the terminal and methine olefinic (C5 and C4)peaks of the starting material 21a were absent.

The solution phase dimerisation of the allylglycine unit 21a isanalogous to ring closing metathesis of allylglycine sidechains in apeptide (Step 1, Scheme 4.1). In order to regioselectively constructmultiple dicarba bonds within a peptide, via the strategy shown inScheme 4.1, the dienamide 57 must not react under the conditions usedfor cross metathesis of allylglycine units 21a (Scheme 4.8).

The dienamide 57 was therefore subjected to analogous dimerisationconditions to those described above for allylglycine 21a. ¹H n.m.r.spectroscopy confirmed complete recovery of the starting olefin 57 withno evidence of the dimerised dienoate 61 (Scheme 4.9). These resultswere very encouraging and supported our postulate that the dienamide 57would be electronically compromised and therefore inert to metathesis(Step 1, Scheme 4.1).

Subsequent asymmetric hydrogenation of the dienoate 57 would activatethe olefin to metathesis by producing a reactive allylglycine unit 21a(Step 2, Scheme 4.1). This hydrogenation proceeds with excellentstereoselectivity (>95% e.e.) and regioselectivity (<3% over-reduction)(Section 4.1.1) as it relies on chelation of the asymmetricRh(I)-catalyst to the enamide olefin and amide carbonyl group. Theterminal C═C bond does not chelate to the catalyst and is therefore notreduced under these conditions. Similarly, the newly formed C═C bond,generated via cross metathesis in Step 1, would be inert to thiscatalyst.

4.1.3 Dimerisation of an Allylglycine Unit in the Presence of anUnsaturated Dimer

In our strategy, the next step involved ring closing metathesis ofallylglycine units in the presence of an unsaturated carbocycle (Step 3,Scheme 4.1).The solution phase model study therefore required thedimerisation of allylglycine in the presence of an unsaturated dimer(Scheme 4.10). A differentially protected allylglycine derivative 62 wassynthesised to facilitate unambiguous assessment of cross metathesisselectivity.

4.1.3.1 Synthesis of (2S)-Methyl 2-N-Benzoylaminopent-4-enoate 62

The benzoyl-protected allylglycine derivative 62 was prepared viacatalytic asymmetric hydrogenation of the dienamide 63. Thehydrogenation precursor 63 was synthesised by Horner-Emmons olefinationof the phosphonate ester 64 which was isolated in three steps fromcommercially available benzamide 35 and glyoxylic acid 41 (Scheme 4.11).

A mixture of commercially available benzamide 35 and glyoxylic acid 41was heated at reflux in acetone to give pure N-benzoyl-2-hydroxyglycine65 as a colourless solid in quantitative yield (Scheme 4.12). The ¹Hn.m.r. spectrum supported formation of the α-hydroxyglycine derivative65 with the appearance of a methine (H2) doublet and broad amide (NH)doublet at δ 5.60 and δ 9.26 respectively. Spectroscopic data were inagreement with those reported in the literature.²⁰⁹

Treatment of N-benzoyl-2-hydroxyglycine 65 with a catalytic amount ofconcentrated sulfuric acid in methanol furnished methylN-benzoyl-2-methoxyglycinate 66 in 87% yield (Scheme 4.13). Thesereaction conditions converted the carboxylic acid to the methyl esterand the hydroxyl functional group to the methyl ether.

The presence of two new methoxyl peaks in the ¹³C n.m.r. spectrum at δ53.2 and δ 57.0 and the corresponding methyl singlets in the ¹H n.m.r.spectrum at δ 3.54 and δ 3.85 supported formation of the desired product66. Spectroscopic data were in agreement with those reported in theliterature.²⁰⁹

Reaction of methyl N-benzoyl-2-methoxyglycinate 66 with phosphoroustrichloride and trimethyl phosphite in toluene at 70° C. gave thephosphonate ester 64 in 76% yield (Scheme 4.14). The appearance of amethine doublet of doublets (H2) at δ 5.47 was consistent with vicinalphosphorous coupling and was in agreement with data reported in theliterature.²¹⁰

(2Z)-Methyl 2-N-benzoylaminopenta-2,4-dienoate 63 was synthesised byHorner-Emmons olefination of methyl2-N-benzoylamino-2-(dimethoxyphosphinyl)acetate 64 with commerciallyavailable acrolein 58 in the presence of tetramethylguanidine (TMG)(Scheme 4.15). The reaction proceeded through a nucleophilicintermediate 67 which reacted with acrolein 58 to afford the dienoate 63as colourless needles in 80% yield.

The ¹H n.m.r. spectrum displayed a new terminal olefin doublet ofdoublets at δ 5.50 and δ 5.64 corresponding to H5-E and H5-Zrespectively in addition to a well-defined methine (H4) doublet ofdoublet of doublets at δ 6.56. Spectroscopic data were in agreement withthose reported in the literature.²¹¹

The final step in the synthesis of (2S)-methyl2-N-benzoylaminopent-4-enoate 62 involved asymmetric hydrogenation ofthe dienamide 63.^(†) Use of Rh(I)—(S,S)-Et-DuPHOS under 30 psi H₂ inbenzene gave the allylglycine derivative 62 with excellentenantioselectivit^(‡) (100% e.e., Scheme 4.16). Approximately 7% of theover-reduced product 68 was obtained under these conditions and attemptsto separate allylglycine 62 from 68 were unsuccessful. The contaminatedsample of allylglycine 62 was used in subsequent reactions as thepresence of the inert impurity 68 would not interfere in the catalyticstrategy. ^(†) The benzoyl-protected olefin 62 can also be prepared intwo steps from commercially available L-allylglycine((2S)-2-aminopent-4-enoic acid).^(‡) Asymmetric hydrogenation of thedienamide 63 with Rh(I)—(R,R)-Et-DuPHOS was performed in order tofacilitate enantiomeric excess determination. Chiral GC confirmed thatthe (R)- and (S)-allylglycine derivatives 62 were produced in 100% e.e.

Formation of allylglycine 62 was supported by ¹³C n.m.r. spectroscopywhich showed the replacement of an olefinic methine (C3) peak with a newmethylene signal at δ 36.8 and a methine (C2) peak at δ 52.1.Spectroscopic data were in agreement with those reported in theliterature.²¹²

4.1.3.2 Dimerisation of (2S)-Methyl 2-N-Benzoylaminopent-4-enoate 62

The benzoyl-protected allylglycine unit 62 was quantitativelyhomodimerised under general cross metathesis conditions using Grubbs'catalyst (Scheme 4.17). The loss of ethylene drives the metathesisreaction to completion.

Purification by flash chromatography furnished (2S,7S)-dimethyl2,7-N,N′-dibenzoylaminooct-4-enedioate 69 as a pale brown solid in 82%yield. ¹H n.m.r. spectroscopy supported synthesis of the dimer 69 withthe replacement of terminal olefin peaks by a new methine (H4, 5)triplet at δ 5.49. The accurate mass spectrum also displayed a molecularion plus sodium peak at m/z 461.1695 which is consistent with thatexpected for the molecular formula C₂₄H₂₆N₂NaO₆.

With the benzoyl-protected allylglycine 62 in hand and characterisationof its dimer 69 complete, we attempted the cross metathesis ofallylglycine 62 in the presence of the unsaturated N-acetyl-protectedallylgycine dimer 60 (Step 3, Scheme 4.4).

4.1.3.3 Dimerisation of (2S)-Methyl 2-N-Benzoylaminopent-4-enoate 62 inthe presence of (2S,7S)-Dimethyl 2,7-N,N′-Diacetylaminooct-4-enedioate60

Cross metathesis of allylglycine derivative 62 in the presence ofunsaturated dimer 60 proceeded with Grubbs' catalyst to afford dimer 69(Scheme 4.18). No mixed cross metathesis product 70 was observed.However, use of the more reactive metathesis catalyst, second generationGrubbs' catalyst, did lead to a mixture of cross metathesis products,69, 70 and recovered dimer 60. The complicated ¹H n.m.r. spectrum didnot allow the distribution of products to be quantified but massspectrometry confirmed the presence of homodimers 60 and 69 and themixed cross metathesis product 70.

These results indicated that in a peptide application of this strategy(Step 3, Scheme 4.1), selective cyclisation of the allylglycine unitswill only be successful in the presence of Grubbs' catalyst and the useof the more reactive second generation Grubbs' catalyst must be avoided.With successful completion of Step 3, we moved to the last step of thestrategy (Step 4, Scheme 4.1).

4.1.4 Wilkinson's Hydrogenation of Unsaturated Dimers

The final step in the model sequence involved reduction of theunsaturated dimers 60 and 69 to give the corresponding saturated dicarbabridges 71 and 72. Homogeneous hydrogenation of dimers 60 and 69 withWilkinson's catalyst, Rh(I)(PPh₃)₃Cl, under mild experimentalconditions, gave the saturated diaminosuberic acid derivatives 71 and 72in excellent yields (>99%) (Scheme 4.19). We employed a homogeneouscatalyst in order to facilitate the on-resin application of thishydrogenation which would otherwise be complicated by the more commonlyemployed heterogeneous systems such as palladium on charcoal.

Formation of the saturated dimers 71 and 72 was supported byspectroscopic analysis which displayed new methylene proton (H3, 4) andcarbon (C3, 4) signals in the ¹H and ¹³C n.m.r. spectra respectively.

4.1.5 Dimerisation of Allylglycine 21a in the presence of (2Z)-Methyl2-N-Acetylaminopenta-2,4-dienoate 57

These results looked very promising: We had successfully completed allfour steps in our devised synthesis (Scheme 4.1). However, our attemptsto dimerise allylglycine 21a in the presence of dienamide 57 wereunsuccessful with both first and second generation Grubbs' catalysts(Scheme 4.20). The inclusion of dienamide 57 also hampered dimerisationof benzoyl-protected allylglycine 62 and led to complete recovery of thestarting dienoate 57 and allylglycine unit 62.

¹H n.m.r. binding studies between the catalyst and dienamide 57 (ratioof 1:1) showed that the dienamide 57 rapidly coordinated to theruthenium centre forming a ruthenium-vinylalkylidene complex 73(spectrum a in FIG. 2). Within 60 minutes, complex 73 had diminished anda new ruthenium species 74 was generated (spectrum b in FIG. 2). Thesecond species is postulated to involve coordination of the estercarbonyl group to the ruthenium centre and liberation of atricyclohexylphosphine ligand 75. Formation of complex 74 was completewithin 2 hours and was stable and unreactive over 18 hours (spectrum cin FIG. 2).²¹³ Unfortunately, attempts to isolate this complex 74 wereunsuccessful.

Furthermore, attempts to regenerate the dienamide 57 from theruthenium-carbonyl chelate 74 via reaction with ethyl vinyl ether andformation of the Fischer-type carbene complex,²¹⁴ failed due toconjugate addition of liberated tricyclohexylphosphine 75 to thedienamide substrate 57. This highlighted the sensitivity of acrylate 57to N- and P-based nucleophiles and potential problems that could ariseduring peptide synthesis, where piperidine is routinely used tofacilitate Fmoc-cleavage from residues prior to coupling.

Although the dienamide 57 was unexpectedly reactive to Grubbs' catalyst,the proposed strategy showed potential. Solution phase experiments withSteps 2-4 (Scheme 4.21) were not problematic and indicated that multipledicarba bond formation was indeed feasible via a modified strategy. Thefirst step, however, required revision. We postulated that the presenceof a substituent at the olefinic terminus of the dienamide substratemight impede binding to the metathesis catalyst and therefore allow thering closing metathesis of the more reactive allylglycine sidechains toproceed.

4.2 Revised Strategy

A revised strategy was investigated centering on the use ofnon-proteinaceous, terminally functionalised allylglycine units. Thismodified route involved: i) metathesis of allylglycine units in thepresence of a terminal-phenyl substituted dienamide 76, and ii)subsequent hydrogenation of the dienamide 76 to yield a more reactiveolefin 77 for the second ring closing metathesis (Scheme 4.22). Wepostulated that the presence of a phenyl substituent at the olefinterminus might impede binding of the metathesis catalyst and circumventthe problems experienced in the first strategy. The solution phase modelstudies of this revised strategy therefore commenced with the synthesisof the phenyl-substituted dienamide 76.

4.2.1 Synthesis of (2Z)-Methyl2-N-Acetylamino-5-phenylpenta-2,4-dienoate

The dienamide 76 was prepared according to a procedure by Burk et al.¹¹⁷from a Horner-Emmons olefination of methyl2-N-acetylamino-2-(dimethoxyphosphinyl)-acetate 39 and commerciallyavailable trans-cinnamaldehyde 78 (Scheme 4.23). The phosphonate 39 wasprepared in three steps from commercially available acetamide 34 andglyoxylic acid 41.

The dienamide 76 was isolated as an off-white solid in 74% yield. Massspectrometry supported formation of the dienoate 76 with a molecular ionplus proton peak at m/z 246.2 which is consistent with that expected formolecular formula C₁₄H₁₆NO₃. Spectroscopic data were in agreement withthose reported in the literature.¹¹⁷

4.2.2 Solution Phase Reactions with Dienamide 76

¹H n.m.r. binding studies of a 1:1 mixture of Grubbs' catalyst anddienamide 76 showed no ruthenium-vinylalkylidene formation. Hence, thissuggested that the poor chelating properties of the modified dienamide76 to Grubbs' catalyst should now facilitate high yieldinghomodimerisation of allylglycine 21a into its dimer 60 (Scheme 4.24).

Surprisingly, homodimerisation of 21a to 60 was found to proceed butwith poor conversion (28%). This suggested that the dienamide 76 wasstill capable of influencing the metathesis cycle. Hopeful that thiswould later be rectified through modification of metathesis conditions,we continued to investigate subsequent steps of the proposed strategy.

Rh(I)-DuPHOS-catalysed asymmetric hydrogenation of dienamide 76 undermild conditions (75 psi H₂) gave (2S)-methyl2-N-acetylamino-5-phenylpent-4-enoate 77 in 99% e.e. (Scheme 4.25).Formation of the desired phenyl-substituted enamide 77 was confirmed byspectroscopic analysis which was in agreement with literature data.¹¹⁷

Disappointingly, cross metathesis of 77 using Grubbs' catalyst wasunsuccessful. After 13 hours, ¹H n.m.r. spectroscopy showed noconversion to the desired homodimer 60. Conditions to facilitate therequired cross metathesis were found, however, using a 5 mol % solutionof second generation Grubbs' catalyst in dichloromethane (Scheme 4.26).A modest conversion (44%) to the expected homodimer 60 was achieved. Inspite of this promising result, this chemistry was not investigatedfurther since the requirement for the more reactive second generationGrubbs' catalyst would render the previously formed unsaturatedcarbocycle vulnerable to further cross metathesis. Mixed crossmetathesis products would therefore result (Section 4.1.3.3).

Selective reduction of the first-formed unsaturated carbocycle prior tothe second metathesis reaction would, however, eliminate the chance ofmixed cross metathesis (Step 2, Scheme 4.27). We therefore subjected thephenyl substituted diene 76 to the hydrogenation conditions previouslydeveloped for the hydrogenation of the unsaturated dimer 60.Unfortunately, these conditions resulted in a 1:4 mixture of olefin 77:saturated derivative 79 (Scheme 4.28).

This disappointing result is not without literature precedent. The rateof olefin reduction by Wilkinson's catalyst is profoundly influenced bysteric hindrance about the C═C double bond, but related reductionsinvolving styrene have previously shown that electronic effects overridethese steric effects and that the aromatic substituent enhances the rateof reduction.^(215,216)

4.3 Final Strategy

The failure of this second strategy led to a final revision and thediscovery of a strategy which would enable the selective hydrogenationof an unsaturated carbocycle in the presence of a deactivated butpotentially metathesis-active olefin. We decided to capitalise on theslow reactivity of trisubstituted olefins to Wilkinson's hydrogenationand their reduced reactivity to metathesis. 1,1-Disubstituted olefins,for example, do not undergo homodimerisation and only react with morereactive olefins.^(130,182) This differential reactivity would thereforefacilitate the cross metathesis of allylglycine units and subsequenthydrogenation without interference from the 1,1-disubstituted olefinresidues. A simple transformation then renders the trisubstituted olefinmore reactive to metathesis and facilitates the formation of the secondcarbocycle (Scheme 4.29).

4.3.1 Synthesis of (2S)-Methyl 2-N-Acetylamino-5-methylhex-4-enoate 19

The prenyl olefin 19 was prepared via asymmetric hydrogenation of thecorresponding dienamide 20. The prenylglycine derivative 19 was isolatedin quantitative yield and excellent enantioselectivity (Scheme 4.30).

4.3.2 Reactions with (2S)-Methyl 2-N-Acetylamino-5-methylhex-4-enoate 19

This prenylglycine unit 19 was subjected to the hydrogenation conditionsthat quantitatively reduce the dimer 60 to the saturated analogue 71(Scheme 4.19) and encouragingly, 94% of the starting enamide 19 wasrecovered (Scheme 4.31). This was a very promising result which promptedus to further investigate cross metathesis reactions involving thissubstrate 19. Furthermore, we envisaged that incorporation of this unit19 into a peptide via solid phase peptide synthesis (SPPS) would bestraightforward.

Cross metathesis of allylglycine unit 21a into dimer 60 in the presenceof the prenyl enamide 19 proceeded smoothly with quantitative conversion(Scheme 4.32); the starting prenyl enamide 19 was recovered unchanged.

The reduced reactivity of prenylglycine 19 enabled the dimerisation ofallylglycine 21a and the selective hydrogenation of the resultanthomodimer 60. The next step in the strategy involves activation of thedormant olefin 19 (Step 3, Scheme 4.29). This can be achieved by crossmetathesis with ethylene via a more active ruthenium alkylidene.

The prenyl compound 19 was subjected to ethenolysis to convert it to themore reactive allylglycine derivative 21a (Scheme 4.33). Exposure of 19to 20 mol % of Grubbs' catalyst under an atmosphere of ethylene resultedin the recovery of the starting olefin 19. Use of the more reactive2^(nd) generation Grubbs' catalyst at higher reaction temperature (50°C.) and ethylene pressure (60 psi) still led to only poor conversions to21a (<32%).

We postulated that this result may be due to the unstable nature of thein situ generated ruthenium-methylidene intermediate 48 at elevatedtemperature (50° C.),202-204 or unfavourable competition between therising concentration of terminal olefins and 21a for binding to theruthenium catalyst.²¹⁷

In order to circumvent this problem, the prenyl enamide 19 was insteadexposed to an atmosphere of cis-2-butene (15 psi) thereby facilitatingthe catalysis via the more stable ruthenium-ethylidene complex 49.Butenolysis of 19 in the presence of 5 mol % second generation Grubbs'catalyst gave the expected crotylglycine derivative 81 with quantitativeconversion (Scheme 4.33).

(2S)-Methyl 2-N-acetylaminohex-4-enoate 81 was isolated as a brown oilin 84% yield after flash chromatography. The ¹H n.m.r. spectrum showedthe replacement of the olefinic methine (H4) triplet in the startingprenyl compound 19 with new olefinic methine (H4, 5) multiplets at δ5.49 and δ 5.24 respectively. Spectroscopic data were also in agreementwith those reported in the literature.^(117,119)

Interestingly, the purity of the 2-butene was found to be critical tothe success of the cross metathesis reaction. When butenolysis reactionswere conducted with a less expensive, commercially available mixture ofcis- and trans-2-butene, only a trace of the butenolysis product wasdetected. Gas chromatographic analysis of the isomeric butene mixtureshowed that it was contaminated with 2.6% butadiene while none wasdetected in the pure cis-2-butene.²¹⁸ The addition of butadiene (2%) tocis-2-butene inhibited formation of the butenolysis product while acis+trans mixture (30:70) of 2-butene, free of butadiene,^(†) led toquantitative conversion to the expected cross metathesis product. Theseresults strongly suggested that butadiene was poisoning the metathesiscatalyst. Grubbs et al. have previously reported that butadiene canreact with the ruthenium-benzylidene catalyst to produce a vinylalkylidene which is inactive for acyclic metathesis reactions.²¹⁹ ^(†)The cis+trans-2-butene mixture (30:70)free of butadiene was obtained byisomerisation of cis-2-butene withbenzylidene[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene]dichloro[bis(3-bromo-pyridine)]rutheniumat −5° C.²¹⁸

This activated crotylglycine derivative 81 was readily crossmetathesised to the expected homodimer 60 with 5 mol % of secondgeneration Grubbs' catalyst in dichloromethane (Scheme 4.34).Spectroscopic data were in agreement with those previously reported(Section 4.1.2).

4.3.3 Reaction Sequence

Finally, an equimolar mixture of olefins 62 and 19 was exposed to atandem sequence of the previously described five homogeneous catalyticreactions: i) dimerisation of allylglycine 62, ii) hydrogenation of theresultant homodimer 69, iii) activation of prenylglycine 19, iv)dimerisation of the activated crotylglycine derivative 81 and v)hydrogenation of the resultant homodimer 60. Solvent removal andsubsequent ¹H n.m.r. analysis was performed on the crude product mixtureafter each transformation. The catalytic sequence resulted inquantitative conversion of the reactive substrate in each step andultimately yielded diaminosuberic acid derivatives 71 and 72 as the onlyisolated products in 84 and 70% yield respectively (Scheme 4.35).

4.4 Summary

In conclusion, these model studies demonstrate that through thecombination of homogeneous catalysis and judicious selection ofnon-proteinaceous allylglycine residues of varying reactivity, a highlyefficient, unambiguous and regioselective synthesis of dicarba analoguesof multi-cystine containing peptides may be achievable. The methodologyis also amenable to natural product and polymer synthesis or whereverselective carbon-carbon bond formation is required. Section 6investigates the application of this methodology to synthetic andnaturally occurring peptides.

5.0 A TANDEM METATHESIS-HYDROGENATION STRATEGY FOR THE SELECTIVEFORMATION OF THREE CARBON-CARBON BONDS

The selective formation of multiple dicarba bonds in complex moleculesis a significant synthetic challenge. In section 4, we devised astrategy for a solution phase regioselective synthesis of two dicarbabridges. This chapter describes a catalytic strategy for theregioselective construction of three dicarba bridges in solution byselective and successive metathesis-hydrogenation transformations.

5.1 Proposed Strategy

In the preceding chapter we achieved regioselective C—C bond formationthrough the use of olefinic substrates possessing tuneable reactivityand highly chemo- and stereoselective catalysts. The varying reactivityof allylglycine and prenylglycine units towards metathesis andhydrogenation has been previously described (Chapter 4). We postulatedthat the steric and particularly electronic effects of a prenylglycinedienoate 82 would render it inert to metathesis and Wilkinson'shydrogenation. Two dicarba bridges can therefore be constructed in thepresence of this inert olefin (Scheme 5.1). The diene can then beactivated in two simple steps, the first of which involves a catalyticasymmetric hydrogenation to give optically pure prenylglycine. We havealready demonstrated the facile activation of the prenyl sidechain bycross metathesis with either ethylene or cis-2-butene to give thecorresponding allyl- or crotylglycine′ derivative respectively. Theresultant activated olefin can readily undergo homodimerisation to givean unsaturated dimer which can be reduced to afford the saturateddicarba bridge. The final product mixture would ultimately contain threedifferent diaminosuberic acid derivatives where the selective C—C bondformation would represent the formation of a dicarba analogue of atricystine-containing peptide (Scheme 5.1). In order to validate theproposed strategy we conducted a series of solution phase reactions.

5.2 Solution Phase Model Study

A metathesis triplet 83, 19 and 82 was developed to facilitate thecontrolled formation of three diaminosuberic acid derivatives (Table5.1). The differing olefin substitution in the molecules providestuneable reactivity towards homogeneous metathesis^(120,121) andhydrogenation catalysts.^(33,215)

TABLE 5.1 Reaction Sequence for the Construction of Three DicarbaBridges^(a)

Step 1: CM-H Grubbs' catalyst Step 2: Wilkason's Hydrogena- tion Step 3:CM 2^(nd) gen. Grubbs' catalyst Step 4: CM-H 2^(nd) gen. Grubbs'catalyst Step 5: Wilkinson's Hydrogena- tion Step 6: Rh(I)- DuPHOSHydrogena- tion Step 7: CM 2^(nd)gen. Grubbs' catalyst Step 8: CM-H2^(nd) gen. Grubbs' catalyst Step 9: Wilkinson's Hydrogena- tion

Substrates C═C C—C Act C═C C—C Act Act C═C C—C Products

Sidechain Reactivity Summary of Activity

✓ ✓ — — — — — — — Terminal allylic olefin. No activation required.

X X ✓ ✓ ✓ — — — — Trisubstituted olefin. Activated via CM with 2-butene.

X X X X X ✓ ✓ ✓ ✓ Hindered extended acrylamide olefin. Activated via i)asymmetric hydrogenation and ii) CM with 2-butene. ^(a) ✓= Reactiveolefin, X = Unreactive olefin, — = Unreactive dicarba bridge, Act =Olefin activation step, CM-H = Cross metathesis-homodimerisation, CM =Cross metathesis

Three different N-acyl protecting groups were employed to facilitateunambiguous assessment of cross metathesis selectivity. A mixture of ap-nitrobenzoyl-protected allylglycine derivative 83, an acetyl-protectedprenylglycine unit 19 and a benzoyl-protected prenylglycine dienamide 82gave adequate separation of characteristic peaks in the ¹H n.m.r.spectrum (FIG. 3) to enable reaction monitoring of Steps 1-9.Importantly, the protecting groups on the amino group do not affect themechanistic course of the reaction sequence.

The solution phase studies therefore commenced with preliminaryexperiments on the diene 82 to ensure it was inert to metathesis andWilkinson's hydrogenation.

5.2.1 Synthesis of (2Z)-Methyl2-N-Benzoylamino-5-methylhexa-2,4-dienoate

The dienamide 82 was synthesised by Horner-Emmons olefination of methyl2-N-benzoylamino-2-(dimethoxyphosphinyl)acetate 64 with commerciallyavailable 3-methyl-2-butenal 40 and tetramethylguanidine (TMG) (Scheme5.2), as described for several dienamides in this application

Methyl 2-N-benzoylamino-5-methylhexa-2,4-dienoate 82 was isolated as anoff-white solid in 73% yield. Formation of the prenylglycine dienamide82 was supported by ¹³C n.m.r. spectroscopy which displayed new olefinicmethyl peaks at δ 19.3 and δ 27.1 respectively, in addition tocharacteristic olefinic methine (C3, 4) and quaternary (C2, 5) peaks. Amolecular ion plus proton peak at m/z 260.1282 in the accurate massspectrum was consistent with the molecular formula C₁₅H₁₈NO₃ and alsosupported formation of the dienamide 82.

5.2.2 Reactivity of (2Z)-Methyl2-N-Benzoylamino-5-methylhexa-2,4-dienoate 82 toward Metathesis andHydrogenation

The dienamide 82 was subjected to homodimerisation conditions withsecond generation Grubbs' catalyst (Scheme 5.3). ¹H n.m.r. spectroscopyconfirmed complete recovery of the starting olefin 82 with no evidenceof the dimerised dienoate 84. This result supported our postulate thatdiene 82 is electronically and sterically compromised and thereforeinert to metathesis.

Our proposed reaction sequence then required the reduction of anunsaturated dicarba bridge in the presence of a diene moiety (Step 2,Scheme 5.1). The dienamide 82 was therefore subjected to thehydrogenation conditions that quantitatively reduce unsaturated dimersto their saturated analogues (Wilkinson's catalyst, 50 psi H₂).Encouragingly, the reduced prenyl compound 85 was not observed and thestarting olefin 82 was recovered unchanged (Scheme 5.3).

Finally, the diene 82 was exposed to metathesis conditions used toactivate prenylglycine 19 by conversion to the crotyl derivative 81(cis-2-butene, second generation Grubbs' catalyst, Scheme 5.3). Again ¹Hn.m.r. spectroscopy indicated that the dienamide 82 was inert to theseconditions. The starting olefin 82 was recovered unchanged with noevidence of the potential cross metathesis product 86.

5.2.3 Activation of (2Z)-Methyl2-N-Benzoylamino-5-methylhexa-2,4-dienoate

Activation of the dienamide 82 was initiated with aRh(I)-Et-DuPHOS-catalysed asymmetric hydrogenation to give theprenylglycine derivative 87 in excellent yield and enantioselectivity(100% e.e.) (Scheme 5.4).

The replacement of olefinic methine (H3, 4) proton peaks in the ¹Hn.m.r. spectrum with new methylene (H3) and olefinic (H4) multiplets atδ 2.52-2.76 and δ 5.08 confirmed formation of the prenylglycine residue87. Over-reduction of the terminal double bond was not observed underthese conditions.

The second activation step involved treatment of the prenyl olefin 87with 5 mol % second generation Grubbs' catalyst and cis-2-butene (15psi) to yield the crotylglycine derivative 88 (Scheme 5.4). The reactionproceeded with quantitative conversion as indicated by ¹H n.m.r. and ¹³Cn.m.r. spectroscopic analysis. The accurate mass spectrum also displayeda molecular ion plus proton peak at m/z 248.1284 which is consistentwith that expected for the molecular formula C₁₄H₁₈NO₃.

5.2.4 Reactions with (2S)-Methyl 2-N-(p-Nitrobenzoyl)aminopent-4-enoate83

The third olefin in the metathesis triplet is the allylglycinederivative 83. Reaction of the hydrochloride salt of allylglycine methylester 51 with p-nitrobenzoyl chloride 89 and triethylamine in a mixtureof dichloromethane:diethyl ether gave the protected allylglycine residue83 in 99% yield (Scheme 5.5).

The ¹H n.m.r. and ¹³C n.m.r. spectra supported formation of theprotected allylglycine 83 with the downfield shift of the methine (H2)doublet of triplets at δ 4.90 and the introduction of aromaticresonances at δ 7.95 (H2′,6′) and δ 8.30 (H3′,5′).

The allylglycine derivative 83 was quantitatively dimerised with Grubbs'catalyst in dichloromethane heated at reflux (Scheme 5.6). Formation ofthe dimer 90 was supported by ¹H and ¹³C n.m.r. spectroscopic analysiswhich displayed signals due to the new olefinic methine proton (H4, δ5.49-5.53) and carbon (C4, 128.8) respectively.

The unsaturated dimer 90 was subjected to the previously describedWilkinson's hydrogenation conditions (50 psi H₂, benzene, 4 hours).Unfortunately, under these conditions, the aromatic nitro substituentswere reduced, thus providing a potential mechanism for poisoning of themetathesis catalyst. Fortunately, Jourdant et al. recently reported theselective reduction of an olefin in the presence of an aromatic nitrogroup.²²⁰ Homogeneous hydrogenation under 15 psi H₂ in a mixture oftetrahydrofuran:tert-butanol (1:1) led to the selective reduction of theunsaturated dimer 90 without concomitant reduction of pendant aromaticnitro groups (Scheme 5.6).

(2S,7S)-Dimethyl 2,7-N,N′-di(p-nitrobenzoyl)aminooctanedioate 91 wasisolated as an off-white solid in 67% yield. The replacement of olefinicpeaks in the ¹H n.m.r. spectrum with new methylene (H4, 6 and H3, 5)multiplets at δ 1.39-1.54 and δ 1.74-2.04 respectively confirmedformation of the saturated dimer 91.

5.2.5 Reaction Sequence

An equimolar mixture of olefins 83, 19 and 82 was subjected to thecatalytic sequence outlined in Scheme 5.7. Solvent removal andsubsequent ¹H n.m.r. and mass spectral analysis was performed on thecrude product mixture after each transformation. Exposure of theolefinic mixture 83, 19 and 82 to Grubbs' catalyst in dichloromethaneled to homodimerisation of allylglycine 83 to form an unsaturateddicarba bridge 90. Predictably, the more sterically hindered olefin 19and the electronically compromised olefin 82 were unreactive under thesereaction conditions. The resultant alkene 90 was then selectivelyhydrogenated in a mixture of tert-butanol:tetrahydrofuran (1:1) withWilkinson's catalyst to afford the saturated dicarba bridge 91. Again,olefins 19 and 82 were inert to these conditions. Both the metathesisand hydrogenation reactions proceeded under mild experimental conditionswith quantitative, unambiguous conversion to give the first suberic acidderivative 91 as shown by n.m.r. and MS analysis.

The next reaction in this catalytic sequence involved the activation ofthe dormant prenyl olefin 19 via cross metathesis with cis-2-butene(butenolysis) to generate a more reactive crotylglycine derivative(Section 4.3.2). The mixture of 91, 19 and 82 was exposed to anatmosphere of cis-2-butene (15 psi) in the presence of 5 mol % secondgeneration Grubbs' catalyst to afford the expected crotylglycinederivative 81 and a trace of the corresponding homodimer 60. Theactivated olefin 81 was then quantitatively homodimerised to theexpected unsaturated dimer 60 with 5 mol % of second generation Grubbs'catalyst. Exposure of the newly formed olefin 60 to a hydrogenatmosphere and Wilkinson's catalyst resulted in quantitative conversionto the saturated dicarba bridge 71 (Section 4.1.4). Once again, thesterically and electronically compromised olefin 82 remained a spectatorover the three reactions used to form the second diaminosuberic acidderivative 71.

The remaining acrylate-type olefin 82 was then used to form the finaldicarba bridge. A double activation sequence was employed to render thisremaining olefin reactive to homodimerisation. Homogeneous hydrogenationof dienamide 82 using chiral Rh(I)—(S,S)-Et-DuPHOS catalyst gave(S)-configured prenylglycine derivative 87 in excellentenantioselectivity (100% e.e.), chemoselectivity and conversion. Noevidence of over-reduction of the C4 carbon-carbon double bond wasobserved. The resulting prenyl olefin 87 was then converted to thecrotylglycine analogue 88 via butenolysis. Exposure of this olefin tothe previously described cross-metathesis and hydrogenation conditionsthen led to the formation of the final dicarba bond and the thirddiaminosuberic acid derivative 72 via alkene intermediate 69. Themetathesis-hydrogenation sequence led to generation of threediamidosuberic acid esters 91, 71 and 72 in 67, 81 and 70% yieldsrespectively. Significantly, residual catalyst and/or decompositionproducts did not compromise subsequent transformations and no otherbyproducts were isolated. This demonstrates the high chemoselectivityexhibited by each catalytic step.

5.3 Summary

A combination of homogeneous hydrogenation and metathesis reactions hasenabled the highly efficient, stepwise chemo- and stereoselectiveformation of three identical dicarba C—C bonds in three different2,7-diaminosuberic acid derivatives without purification ofintermediates. This homogeneous catalytic methodology can be used widelyin peptidomimetics and total product synthesis where multiple(preferably 3) C—C bonds and/or rings need to be selectivelyconstructed.

6.0 Synthesis of Dicarba Cyclic Peptides Via Regioselective CrossMetathesis

This section describes the application of the regioselective strategydeveloped in section 4 to a series of peptides. A model syntheticpentapeptide was initially investigated. The results from this substrateled to the production of dicarba analogues of conotoxin ImI.

6.1 Solid Phase Peptide Synthesis (SPPS)

Linear peptides were synthesised via standard solid phase peptidesynthesis (SPPS) methodology.²²¹ This procedure involves the attachmentof an N-Fmoc-protected amino acid to a solid support and theconstruction of the sequence from the C- to N-terminus Scheme 6.1.Peptide construction requires: i) Fmoc-deprotection of theresin-tethered amino acid under basic conditions, ii) activation of theincoming Fmoc-protected amino acid and iii) its subsequent coupling tothe resin-tethered amino acid. The process is repeated until the desiredpeptide sequence is constructed. Conveniently, the use of orthogonallyprotected amino acids enables sequential Fmoc-deprotection and couplingwithout loss of acid-sensitive sidechain protecting groups.

The choice of resin plays an important role in peptide synthesis. Aplethora of polystyrene-based supports are commercially available. Theseresins are typically cross-linked polystyrene (PS) containing 1%divinylbenzene and are functionalised with linkers (or handles) toprovide a reversible linkage between the synthetic peptide chain and thesolid support.²²¹ Several linkers commonly utilised in Fmoc-SPPS arepresented in. Diagram 6.1. With the target peptide in mind, theappropriate resin-linker can be chosen to functionalise the C-terminusas a carboxylic acid, carboxamide, ester or alcohol. In addition,peptides can be cleaved under acidic or basic conditions where acidsensitive sidechain protecting groups can be retained or simultaneouslydeprotected during peptide cleavage. Importantly, the resin-linkers mustbe inert to metathesis and hydrogenation catalysis conditions.

Construction of the linear peptides via solid phase methodology providestwo options for the construction of dicarba bridges: The complete linearsequence can be cleaved from the resin and then subjected to metathesisand hydrogenation in solution. Alternatively, the regioselectivecatalytic sequence can be performed entirely on the resin-boundpeptides.

We have conducted an on-resin metathesis-hydrogenation sequence for thepreparation of carbocyclic analogues of cystine-containing peptides.This strategy involves conventional solid phase peptide synthesisfollowed by on-resin ruthenium-catalysed ring closing metathesis andon-resin homogeneous rhodium-catalysed hydrogenation of the resultantunsaturated bridge (Scheme 6.2).

The on-resin strategy, however, is compromised by decreased activity ofthe metathesis and hydrogenation catalysts in the heterogeneous system.Previous studies have shown that higher catalyst loadings and longerreaction times are required to achieve quantitative conversion onresin-bound substrates.^(141,142)In addition, ring closing metathesis ofpeptidic substrates is highly sequence dependent due to the involvementof aggregation phenomena. We have found that peptide aggregation,resulting from interchain secondary structures, can lead to poorsolvation of the peptidyl-resin, reduced reagent penetration andultimately low reaction yields. Strategies had to be developed toaddress these problems.

6.2 Ring Closing Metathesis Reactions of Synthetic Pentapeptides

We have investigated the synthesis of bis-dicarba analogues of bicyclicpeptides possessing two disulfide bonds. To achieve this aim we requiredthe use of complimentary pairs of both allyl- and prenylglycine residues(although variations described above can be used). In order to transferthe solution phase methodology across to the solid phase, we needed todemonstrate that Fmoc-protected prenylglycine 92 could be i) synthesisedand incorporated into a peptide sequence using standard SPPS protocol;that it was stable to peptide coupling and deprotection conditions, andiii) that it possessed analogous reactivity to its solution phasecongener in the catalysis steps. We therefore decided to synthesisemodel peptides based on naturally occurring conotoxinpeptides.^(171,172,225) Conotoxin ImI 93 (Ctx ImI) is a smalldodecapeptide possessing two cystine bonds.^(173,174,226) A truncatedsequence 94 of the Cys8-Ala9-Trp10-Arg11-Cys12 Ctx ImI domain wasinitially investigated. This sequence possesses two allylglycineresidues which undergo ring closing metathesis to yield an unsaturatedcarbocycle 95. After establishing optimum conditions for the formationof the first dicarba bond, the sequence was modified to include aprenylglycine residue to facilitate the formation of a second dicarbalinkage.

The pentapeptide 94 was synthesised on Rink amide resin, apolystyrene-based solid support bearing an amine linker that generates aC-terminal carboxamide upon resin cleavage. (Diagram 6.1). Prior toattachment of the first amino acid, the resin was swollen indichloromethane to increase surface availability of resin active sitestowards the incoming C-terminal Fmoc-protected amino acid. Peptideconstruction began with attachment of non-proteinaceousFmoc-L-allylglycine (Fmoc-Hag-OH) 96 to Rink amide resin (A, Diagram6.1) and remaining resin active sites were capped with acetic anhydride.Fmoc-deprotection of resin-tethered allylglycine followed by coupling ofthe successive amino acid and repetition of these steps (B and C,Diagram 6.1) enabled chain elongation. After coupling the last aminoacid, a small aliquot of peptidyl-resin was exposed to trifluoroaceticacid cleavage solution (D, Diagram 6.1) to liberate the peptide 94 as acolourless solid. The mass spectrum displayed a molecular ion peak atm/z 847.1 (M+H)⁺ which was consistent with the formation of thepentapeptide 94.

After confirming that pentapeptide synthesis had been successful, ringclosing metathesis of the fully-protected resin-tethered sample 94a wasperformed with 20 mol % Grubbs' catalyst in dichloromethane and 10%lithium chloride in dimethylformamide. Mass spectral analysis of acleaved aliquot of peptide indicated that these conditions resulted incomplete recovery of the linear peptide 94. Use of the more activesecond generation Grubbs' catalyst did, however, lead to unsaturatedcarbocycle 95 but cyclisation failed to go to completion (Scheme 6.3).The presence of molecular ion peaks at m/z 819.2 (M+H)⁺ and m/z 847.2(M+Na)⁺ were consistent with the presence of the unsaturated carbocycle95 and the linear peptide 94 respectively.

We postulated that the peptide sequence itself may be responsible forthe reduced ring closing metathesis yield. Pentapeptide 94 lacks aproline residue between the two allylglycine sidechains and hence thepredominance of transoid peptide bonds would disfavour a closearrangement of the reacting terminal olefins. The inclusion of turninducers in a peptide sequence can reduce peptide aggregation via theformation of cisoidal amide bonds.²²⁷⁻²²⁹ In addition, the resultantturn can position the reactive allylglycine sidechains in closeproximity to each other and thus facilitate cyclisation. The peptide wastherefore reconstructed to incorporate proline, a naturally occurringturn-inducing amino acid.

The pentapeptide 97 was synthesised on Rink amide resin via the generalSPPS methodology previously described. The peptide possessed an Ala→Proreplacement adjacent to the N-terminal allylglycine residue. Formationof the pentapeptide 97 was confirmed by mass spectrometry with theappearance of a molecular ion peak at m/z 873.2 (M+H)⁺.

Ring closing metathesis of the fully protected resin-bound peptide 97awith Grubbs' catalyst (20 mol %) in dichloromethane and 10% lithiumchloride in dimethylformamide led to recovery of the starting peptide 97with only a trace of product 98 evident in the mass spectrum. Use ofsecond generation Grubbs' catalyst (20 mol %), however, led to completecyclisation (Scheme 6.4). The appearance of molecular ion peaks at m/z845.1 (M+H)⁺ and m/z 867.1 (M+Na)⁺ in the mass spectrum confirmedformation of the unsaturated carbocycle 98. This result clearlydemonstrates the influence of the turn-inducing proline residue onpeptide conformation and reactivity.

In conjunction with this study, we simultaneously assessed the role ofthe catalytic cycle in affecting ring closing metathesis yield. Wepostulated that the incomplete cyclisation of linear sequence 94 couldbe due to thermal decomposition of the ruthenium-methylideneintermediate 48. We therefore investigated synthesis of thecrotylglycine-containing peptide, Fmoc-Crt-Ala-Trp-Arg-Crt-NH₂ 99, forwhich metathesis proceeds through the more stable ruthenium-ethylidenespecies 49.

This initially required the synthesis of the crotylglycine derivative100. Acid-promoted hydrolysis of (2S)-methyl 2-N-acetylaminohex-4-enoate81 gave (2S)-2-aminohex-4-enoic acid hydrochloride salt 101.Fmoc-protection of amino acid 101 was performed according to theprocedure described by Paquet et al. usingN-fluorenylmethoxycarbonylamino-suecinimide (Fmoc-OSu) in aqueous sodiumcarbonate and acetone (Scheme 6.5).²³⁰

¹H n.m.r. and ¹³C n.m.r. spectral analysis of the product confirmed theformation of (2S)-2-N-fluorenylmethoxycarbonylaminohex-4-enoic acid(Fmoc-Crt-OH) 100 with the downfield shift of the methine proton (H2)peak (δ 4.55) and the corresponding carbon signal (δ 52.3). In addition,the appearance of aromatic signals characteristic of the Fmoc-groupsupported product formation. Spectroscopic data were also in agreementwith those reported in the literature.¹⁴⁶

With the Fmoc-protected crotylglycine derivative 100 in hand, wesynthesised the linear peptide, Fmoc-Crt-Ala-Trp-Arg-Crt-NH₂ 99, on Rinkamide resin using the SPPS methodology previously described. The massspectrum displayed a molecular ion peak at tn/z 875.2 (M+H)⁺corresponding to the linear peptide 99.

Ring closing metathesis of the linear resin-tethered peptide 99a withsecond generation Grubbs' catalyst (20 mol %) in dichloromethane and 10%lithium chloride in dimethylformamide led to quantitative formation ofthe unsaturated carbocycle 95^(\) (Scheme 6.6). Note: RCM of thecrotylglyeine-containing peptide 99 leads to the same unsaturatedcarbocycle 95 resulting from cyclisation of the allylglycine-containingsequence 94, i.e. Fmoc-c[Hag-Ala-Trp-Arg-Hag]-OH is identical toFmoc-c[Crt-Ala-Trp-Arg-Crt]-OH.

These studies revealed two successful strategies for the synthesis of adicarba cyclic peptide: i) the inclusion of proline residues to induce aturn in the peptide backbone and ii) the use of crotylglycine to avoid aruthenium-methylidene intermediate in the catalytic cycle. Manynaturally occurring cyclic peptides possess proline residues in theirprimary sequences and this could be used to advantage in RCM reactions.On the other hand, if the target peptide does not possess a prolineresidue (or a residue which can temporarily act as a pseudo-proline),incorporation of a non-native proline residue to enhance RCM yield islikely to have significant structural and biological impact on the finalpeptide. In this case, the use of crotylglycine residues would bebeneficial.

6.2.1 Synthesis of Dicarba-AOD Using Pseudoproline Residues

The Melbourne-based pharmaceutical company Metabolic have a peptidicagent, AOD9604, currently undergoing clinical trials. AOD9604 143 is apeptide fragment derived from the C-terminus of human growth hormone(hGH) and is believed to be responsible for the lipolytic activity ofhGH.²⁶⁷ This 16-residue peptide was derived from the parent anti-obesitydrug AOD9401 144 by addition of a terminal tyrosine residue, and isknown to induce lipolysis and fat oxidation in vitro in adiposetissue.²⁶⁷ Ng et al. report the synthesis of both of these peptidesusing standard solid phase peptide synthesis techniques.^(267,268)

The x-ray crystal structure of native hGH shows that the region ofinterest (residues 177-191) contains a disulphide bridge betweenresidues 182 and 189. An alanine scan of AOD9401 showed that whencysteine was replaced by alanine a dramatic reduction in antilipogenicactivity was observed.²⁶⁸ This suggests that the cystine bridge and thecyclic conformation of the peptide are vital for the activity of AOD9401and related peptide analogues.²⁶⁸ Thus, we were interested insynthesising the dicarba analogue of AOD9604 using the technologydeveloped and described herein to provide analogues with increasedbiological stability.

6.2.1.1 Synthesis of Linear Hag⁶-Hag¹³ AOD9401 and AOD9604

The linear derivative of the carbocyclic analogue of AOD9401 wasinitially synthesised utilising natural amino acids, as well as thenon-proteinaceous residue allylglycine in place of cysteine. Uponsynthesis of the linear peptide 145, an aliquot was subjected tocleavage conditions to assess the success of the synthesis. Massspectral analysis indicated the synthesis of linear Fmoc-protectedAOD9401.

At this point, it was established that AOD9604 would be a more suitabletarget molecule, and the additional amino acid residue was coupled tothe parent AOD9401 molecule already synthesised. The presence of thelinear Hag⁶-Hag¹³ containing derivative 146 was confirmed by massspectral analysis.

6.2.1.2 Synthesis of Dicarba AOD9604 147

Ring closing metathesis, catalysed by second generation Grubbs' catalystwas employed to achieve cyclisation leading to the synthesis ofunsaturated dicarba AOD9604 147. Initially, standard metathesisconditions were used, as perfected in the synthesis of somatostatinanalogues. Lithium chloride was employed to decrease aggregation and 20mol % catalyst loading was used to initiate the metathesis reaction.Mass spectral analysis post-TFA cleavage indicated the failure ofcyclisation, with the only peaks indicative of linear Fmoc-protectedstarting material 146. This reaction was repeated a number or times,including with a higher boiling solvent, however, all attempts yieldedsolely uncyclised starting material.

Deleterious hydrogen bonding in the linear peptide was suspected as thecause of this failed ring closure under standard metathesis conditions.Hence, microwave-accelerated ring closing metathesis of the sameresin-tethered peptide was attempted. Similar catalytic conditions toprevious attempts were employed, with dichloromethane as the solvent.The temperature was increased from 40° C. to 100° C. and the timedecreased to just 10 h. Again, mass spectral analysis of cleavedmaterial indicated the failure of the reaction.

Attention was turned to the primary sequence of the peptide itself. Itwas identified that residues such as proline and glycine can induceturns in peptides, and thus facilitate N→C cyclisation of peptides.N-alkylated residues and D-amino acids can also achieve this. There is alack of any turn-inducing amino acid residues (peptides) in the sequenceof AOD9604, a potential contributing factor in the failure to cyclise.

6.2.1.3 Incorporation of a Turn-Inducing Pseudoproline Residue

Proline is the only naturally occurring amino acid which is known toinduce cis/trans isomerisation about a peptide bond, a feature known toinduce a turn in the peptide backbone, often resulting in a reversal ofthe direction of the backbone. This has led researchers to developalternatives to native proline, and numerous mimetics which produceproline-like cis-peptide bonds and reverse turns have beeninvestigated.²⁶⁹

Pseudoproline (ψPro) residues derived from naturally occurring serine,threonine and cysteine residues have gained popularity in recent years.Their formation is reversible; they are synthesised by acyclocondensation reaction with an aldehyde or ketone and upon exposureto acidic conditions they revert to the parent amino acid.

The incorporation of pseudoproline residues into peptide sequencesincrease the rate and yield of head to tail cyclisation(macrolactamisation). It was decided to incorporate a pseudoprolineresidue in the synthesis of the linear AOD analogue and to conduct themetathesis under microwave irradiation conditions. There are two serineresidues in the sequence, and serine 13 was chosen to be replaced by apseudoproline residue. The incorporation of a pseudoproline residue ishighly dependent on the adjacent residue attached to the amine of thepseudoproline. Pseudoprolines are incorporated into the peptide sequenceas a dipeptide due to the ease of synthesis and stability. Adjacent toserine 9 is an arginine residue; this pseudoproline is not commerciallyavailable and is highly difficult to synthesise due to the bulky sidechain and equally bulky protecting group necessary for peptidesynthesis.

The linear peptide was again synthesised, this time with the dipeptidesequence -Ser(^(t)Bu)-Gly-replaced with the commercially availablepseudoproline analogue. This residue reverts to the required dipeptideupon exposure to the acidic cleavage solution after the cyclisationstep.

The microwave-accelerated metathesis reaction was repeated using theresin-tethered, pseudoproline-containing peptide 146a. After 1 h, analiquot of resin was exposed to cleavage conditions. Mass spectralanalysis indicated the reaction had been successful, with the presenceof a peak at m/z 1000.1 corresponding to the doubly charged adduct ofthe unsaturated dicarba product 147. This example clearly illustratesthe importance of using turn-inducing residues when the metathesisablegroups are not naturally proximate to facilitate high yielding ringclosure.

Finally, the carbocyclic peptide 147 was obtained with a 75% conversionfrom the linear parent moiety 146a. A large aliquot of resin was exposedto cleavage conditions, and purification via preparative HPLC yieldedthe desired peptide in 6% yield. The low yield was attributed topurification difficulties caused by lingering catalyst, despitetreatment with DMSO prior to cleavage, a technique thought to destroyinteraction between the catalyst and resin.

Catalytic hydrogneation of the unsaturated AOD peptide 147 proved to bedifficult. Exposure of the peptide to Wilkinson's catalyst and 90 psi ofhydrogen for 4 days failed to achieve complete reduction of the peptideto the saturated AOD derivative 148. The two dicarba analogues 147 and148, however, were readily separated from each other using preparativeHPLC.

6.3 Regioselective Synthesis of an Intra- and Intermolecular DicarbaBridge in a Synthetic Pentapeptide

Capitalising on the findings of the previous study (Section 6.2) weconstructed another model peptide, Fmoc-Hag-Pro-Pre-Arg-Hag-OH 102, witha strategically placed proline residue. The synthetic pentapeptide 102contains two types of metathesis active groups: Two allylglycine (Hag)residues and a less reactive prenylglycine unit (Pre). This linearsequence facilitates the regioselective construction of two dicarbabonds: An intramolecular metathesis reaction (RCM) of the allylglycineresidues generates a carbocyclic ring and the remaining prenylglycinecan be used to form an intermolecular dicarba bridge via crossmetathesis (CM) with a second unsaturated molecule (Scheme 6.7).

This second dicarba linkage could be used to attach the carbocyclicpeptide to another peptide chain, a drug molecule, a solid support or achelating heterocycle for the generation of radiopharmaceuticals.

Synthesis of the peptide 102 firstly required the preparation of theFmoc-protected prenylglycine derivative (Fmoc-Pre-OH) 92. Crossmetathesis of Fmoc-protected allylglycine 96 with 2-methyl-2-butene inthe presence of 5 mol % second generation Grubbs' catalyst gave thetarget (2S)-2-N-fluorenylmethoxycarbonylamino-5-methylhex-4-enoic acid92 with quantitative conversion (Scheme 6.8).

¹H n.m.r. spectroscopy confirmed formation of the trisubstitutedolefinic amino acid 92 by the replacement of terminal olefinic peakswith a new methine multiplet (H4) at δ 5.11 and two methyl singlets at δ1.63 and δ 1.73. These signals are consistent with the generation of aprenyl group. The accurate mass spectrum also displayed a molecular ionpeak at m/z 388.1525 (M+Na)⁺ which was consistent with that required for92. Unfortunately, purification of the product 92 from residual catalystwas difficult. We later found, however, that the crude amino acid 92could be used without affecting subsequent SPPS procedures.

The peptide 102 was synthesised on inexpensive, readily available Wangresin, a polystyrene-based solid support bearing a benzylic alcohollinker (FIG. 6.1). The non-proteinaceous prenylglycine residue 92 wasincorporated into the peptide sequence without complication. Formationof the pentapeptide 102 was confirmed by mass spectral analysis with theappearance of a molecular ion peak at m/z 813.5 (M+H)⁺ and an additionalpeak at m/z 831.5 (M+H₂O+H)⁺. The latter peak was due to theacid-promoted hydration of the prenyl sidechain during peptide cleavage,leading to the alcohol 103. The hydration of the prenyl group underacidic conditions was not unexpected. During the acid-catalysedcyclisation of the simple prenylglycine derivative 19 to pseudo-proline18, acid-mediated hydration yielded alcohol 47 as a minor byproduct.

After confirming the synthesis of the pentapeptide 102, thepeptidyl-resin was subjected to the regioselective catalytic strategyoutlined in section 4. This is presented in Scheme 6.7.

The first step involved selective RCM of the allylglycine residues inthe presence of the less reactive prenyl sidechain. RCM of theresin-tethered pentapeptide 102a was performed with 40 mol % secondgeneration Grubbs' catalyst in dichloromethane and 10% lithium chloridein dimethylformamide and, as expected, incorporation of prenylglycinedid not hinder cyclisation (Scheme 6.9). Mass spectral analysis of acleaved aliquot of peptide confirmed formation of the unsaturatedcarbocycle 104 with the appearance of a molecular ion peak at m/z 785.4(M+H)⁺. A peak at m/z 803.4 (M+H₂O+H)⁺, corresponding to a hydratedprenyl sidechain in the cyclic product, was also evident. Importantly,prenylglycine remained inert to the metathesis conditions and no mixedcross metathesis products were observed.

Attempts to decrease reaction time and catalyst loading led toincomplete reaction. We therefore decided that the high catalyst loadingand extended reaction times could be tolerated in order to avoid thetime consuming and poor yielding HPLC purification of mixtures resultingfrom non-quantitative cyclisation reactions. Decreasing peptide loadingon the resin (from 0.9 to 0.3 mmolg⁻¹) did, however, enable complete RCMwith 10 mol % of second generation Grubbs' catalyst. This is probablydue to the fact that the use of low substitution resins decreases thedensity of peptide chains on the solid phase and minimises aggregation.The reduced loading enhances resin solvation and reagent access andultimately leads to improved reaction yields.

Selective hydrogenation of the resin-bound unsaturated carbocycle 104awas performed under 80 psi of hydrogen with homogeneous Wilkinson'scatalyst, Rh(I)(PPh₃)₃Cl, in a mixture of dichloromethane:methanol (9:1)(Scheme 6.10). This solvent system served a dual function in maintaininga swollen resin (dichloromethane) and participating in the catalyticcycle (methanol). After 22 hours, a small aliquot of peptide was cleavedand analysed by mass spectrometry. The appearance of peaks at m/z 787.3(M+H)⁺ and m/z 805.4 (M+H₂O+H)⁺ were consistent with formation of thesaturated carbocycle 105. Importantly, the prenyl group remained stableto these reducing conditions which was consistent with the observedreactivity of prenylglycine in the solution phase model studies.

So far, the application of the solution phase methodology to resin-boundpeptide substrates was proceeding as expected. A need for longerreaction times and catalyst loadings was apparent, however, andhighlighted the subtle differences between the two approaches. Afterselective ring closing metathesis, the remaining prenylglycine residuewas employed for the formation of the second dicarba bond.

Activation of the prenyl group was achieved via butenolysis of theresin-bound pentapeptide 105a. The peptide was exposed to an atmosphereof cis-2-butene (15 psi) and 40 mol % second generation Grubbs' catalystin dichloromethane for 42 hours. This led to a mixture of the desiredproduct 106 and the starting peptide 105. The reaction was unexpectedlyand inexplicably slow compared to the analogous solution phaseactivation step. The recovered resin-peptide was therefore re-subjectedto analogous butenolysis conditions which led to the formation of thetarget crotylglycine-containing peptide 106 (Scheme 6.11). Mass spectralanalysis of the cleaved peptide displayed the product molecular ion peakat m/z 773.2 (M+H)⁺ and no evidence of the startingprenylglycine-containing peptide 105 was observed.

2-Butene

High purity 2-butene was found to be critical for high turnovers inbutenolysis reactions (when butane is the disposable olefin). Forexample, when butenolysis reactions were conducted on an unsaturatedtriglyceride (triolein) with commercially available and less expensivecis+trans-2-butene mixtures only traces of butenolysis products weredetected, even with high catalyst loadings. GC analysis of the isomeric2-butene mixture showed that it was contaminated with 2.6% of butadiene,while none of this impurity was found in the commercially availablecis-2-butene. The addition of 1,3-butadiene (2%) to pure cis-2-butenegave a mixture that did not give cross-metathesis products with trioleinwhile a cis+trans-2-butene mixture (30:70) free of 1,3-butadiene wasfound to give the same activity in butenolysis reactions as purecis-2-butene. These results suggested that the 1,3-butadiene was actingas a poison in reactions employing commercial grade cis+trans-2-butene.This discovery is significant and previously unreported; a GC trace ofcommercially available trans 2-butene is contaminated with 1,3-butadiene(FIG. 5), GC traces of cis+trans-2-butene mixtures show the sameimpurities. In conclusion, cis-, trans- and mixtures ofcis+trans-2-butene can all be used in butenolysis (unblocking reactionswith a disposable olefin) reactions but all must be 1,3-butadiene free.Later work with other disposable olefins shows that functionalisation ofthe C1 or C4 carbon atoms of 2-butene further improves turnover,especially for resin-based peptides.

A cross metathesis reaction between the activated-resin bound peptide106a and crotylglycine derivative 81 was then performed. We decided toinvestigate microwave technology as a means of decreasing reaction timein the solid-phase approach. Microwave irradiation of a mixture ofresin-tethered peptide 106a with 40 mol % second generation Grubbs'catalyst, excess crotylglycine 81 (˜50 equiv) in dichloromethane and 10%lithium chloride in dimethylformamide resulted in formation of thedesired intermolecular dicarba linkage (Scheme 6.12). Mass spectrometryconfirmed product formation 107 with the appearance of a molecular ionpeak at m/z 902.3 (M+H)⁺.

Wilkinson's hydrogenation of the unsaturated intermolecular bridge wasachieved under conditions previously established (80 psi H₂,dichloromethane:methanol (9:1), room temperature, 22 hours) to give thetarget peptide 108 containing two regioselectively constructed dicarbabridges (Scheme 6.13).

The successful application of the solution phase methodology (section 4)to a resin-bound pentapeptide 102a led to selective construction of anintramolecular and an intermolecular dicarba bridge. Several importantbiologically active peptides, such as those within the insulinsuperfamily (insulin and relaxin), possess metabolically unstable inter-and intramolecular cystine bonds. This methodology can be applied to theregioselective construction of stable dicarba analogues of thesepeptides. We next examined the extension of this strategy to theconstruction of bicyclic peptides—as cystino-dicarba analogues andbis-dicarba analogues. The latter analogues require the formation of twointramolecular dicarba bridges via sequential ring closing metathesisreactions.

Liskamp et al. recently reported the synthesis of a crossedalkene-bridge of the complex DE-bisthioether ring system of nisin, alantibiotic that possesses five thioether bridges (as distinct todisulfide bridges—which are less stable) (Diagram 6.2).¹⁵⁷

A linear precursor 109 containing four identical allylglycine residueswas subjected to a solution phase double ring closing metathesisreaction. The first cyclisation reaction yielded four out of a possiblesix mono-cyclic peptides. Successive ring closing metathesis undersimilar conditions ultimately yielded the target 1-4, 3-6-carbocyclicpeptide 110 (72%) and a contaminating 1-3, 4-6-bicycle 111 (19%) (Scheme6.14).

These results suggest favourable pre-organisation of the linear peptidefor generation of the target regioisomer.¹⁵⁷ The selective synthesis ofmultiple bridges, however, is rarely so fortuitous.^(129,225,231,232)Indeed, in the synthesis of native conotoxin sequences, severaltopoisomers (ribbon, globule and beads) are obtained after oxidativefolding.^(225,231,233) Multiple cystine formation usually requires anorthogonal protection strategy and sequential oxidation of cysteineresidues.²²⁵ For this reason, we investigated the regioselectivemethodology developed in section 4 for the synthesis of dicarbaanalogues of a native conotoxin sequence, Ctx ImI 93.

6.4 Synthesis of Dicarba Analogues of Conotoxin ImI

Conotoxins are venom components of cone snails (Conidae) and represent agroup of small disulfide-rich peptides that act as potent and highlyspecific antagonists for different receptor targets.¹⁷¹⁻¹⁷⁴ Conotoxinsderive their receptor subtype specificity from the arrangement of theirdisulfide bonds and resultant loop sizes. For example, α-conotoxins,which contain two disulfide bonds in a 1-3, 2-4 arrangement (Diagram6.3), target nicotinic acetylcholine receptors of vertebrates.²³⁴χ-Conotoxins, on the other hand, possess a 1-4, 2-3 disulfide bondarrangement and are selective for noradrenaline transporters.

The small size (typically between 10-40 amino acids), selectivity andpotency of conotoxins make them ideal therapeutic candidates forclinical conditions such as pain, epilepsy, stroke and cancer.^(171,234)Recently Ziconotide, an ω-conotoxin, completed Phase III clinical trialsfor neuropathic pain whilst two new conotoxin analogues (ω-Ctx CVID andχ-Ctx MrIA) are in clinical trials for chronic pain.

Conotoxins possess a rich diversity of amino acid residues and this,coupled with their potential as pharmaceutical agents, makes themchallenging and interesting targets. We chose to examine α-conotoxin ImI93 (Ctx ImI), a small cysteine rich dodecapeptide isolated from thevermivorous conus species Conus imperialis. ^(173,174,226) Its twointramolecular disulfide bonds form the hydrophobic core of the moleculeand generate a constrained two loop structure which, together with acentral proline residue, arrange three essential residues (Asp5, Arg7,Trp10) for selective interaction with complementary residues within theα7 neuronal nicotinic acetylcholine receptor.

Interestingly, the structural and functional role of the disulfide bondsin these natural products is yet to be elucidated. Generation ofdicarba-cystino hybrids of conotoxin ImI and ultimately bis-dicarbaanalogues allows the importance of the constituent bridges on thestructure and activity of the peptide to be elucidated. We thereforeinvestigated the application of the on-resin metathesis-hydrogenationsequence to generate a library of dicarba analogues of conotoxin ImI(Diagram 6.4).

Metathesis catalysts display high functional group tolerance andhomogeneous rhodium-based catalysts, unlike their heterogeneouscounterparts, are not poisoned by sulfur-containing functionality. Wedecided to initiate our study with the synthesis of dicarba-cystinohybrids of Ctx ImI.

6.4.1 Cystino-Dicarba Hybrids of Conotoxin ImI

Native α-conotoxins are amidated at their C-termini. Rink amide resinwas therefore chosen to facilitate linear peptide construction andgenerate the required C-terminal carboxamide upon resin cleavage. Thelow loading (0.52 mmolg⁻¹) of the Rink amide linker helps to reducecrowding and aggregation of peptide chains and reduces the likelihood ofhomodimerisation in the subsequent metathesis reaction. Standard SPPSusing HATU-NMM activation and Fmoc-protected amino acids was used toconstruct the two linear peptides: [2,8]-Hag-[3,12]-Cys conotoxin ImI112 and [2,8]-Cys-[3,12]-Hag conotoxin ImI 113. Both of these sequencespossess two strategically placed non-proteinaceous L-allylglycine (Hag)residues to facilitate construction of the dicarba bridge. Intermediateswere carried through without purification or characterisation up to thedodecapeptides 112 and 113. A sample of each linear peptide was obtainedby cleavage from the resin and determined to be of >95% purity byreverse-phase-HPLC. Mass spectral analysis gave the molecular ion peakat m/z 1565.7 (M+H)⁺ and the corresponding doubly charged ion peak atm/z 783.5 [½(M+2H)]⁺. Both ions are consistent with the structures ofthe isomeric sidechain deprotected linear peptides 112 and 113.

Ring closing metathesis was performed on resin-attached linear peptidesto eliminate any potential problems arising from dimerisation and/orpoor peptide solubility. Exposure of [2,8]-Hag Ctx ImI 112a to firstgeneration Grubbs' catalyst (50 mol %) in dichloromethane at 50° C. for72 hours gave only trace amounts (<10%) of cyclised product 114. Themore reactive second generation Grubbs' catalyst was then used toimprove the cyclisation yield (Scheme 6.15). While RCM progressedfurther (˜70%) with this catalyst, conditions could not be found toeffect full cyclisation to 114. Change in solvent, concentration,catalyst loading, and reaction time had no positive effect onconversion. The addition of a chaotropic salt (lithium chloride indimethylformamide) to the reaction mixture also had no effect on RCMyield. Similarly, RCM of a dicrotylglycine analogue of the primarysequence of 112, which avoids catalytic cycling through an unstableruthenium-methylidene species, also failed to achieve completeconversion to the cyclic target 114.

Construction of the isomeric [3,12]-unsaturated carbocyclic Ctx ImI 115was found to be even more problematic. Exposure of the resin-boundpeptide 113a to both first and second generation Grubbs' catalysts undera variety of experimental conditions failed to yield the unsaturatedcarbocycle 115. Possible reasons for the poor reactivity of this isomer113 included the diminished influence of the proline residue inassisting formation of the larger carbocycle (28-membered ring) and theclose proximity of the C-terminal allylglycine residue to the bulky Rinkamide linker. The sequence was therefore reconstructed on BHA resinbearing a linear HMBA-Gly-Gly-linker. Cyclisation of the BHA resin-boundpeptide was attempted in the presence of 20 mol % second generationGrubbs' catalyst and chaotropic salts. Unfortunately, mass spectralanalysis of the product mixture again showed only the starting peptide113.

Microwave-assisted ring closing metathesis of isomeric linear peptides112a and 113a provided both of the target carbocycles 114 and 115. Inour study, a microwave reactor emitting a focused irradiation at 2.45GHz with a maximum power of 300 W was used. Irradiation of a mixture ofRink amide-bound [2,8]-Hag-[3,12]-Cys Ctx ImI 112a and second generationGrubbs' catalyst (10 mol %) in dichloromethane containing 10% lithiumchloride in dimethylformamide resulted in complete ring closure in onlyone hour (Scheme 6.16). Decreasing the catalyst loading (5 mol %) alsoled to quantitative conversion to the unsaturated carbocycle 114 afterjust two hours of microwave irradiation. Mass spectral analysis of theproduct mixture showed the required molecular ion peak at m/z 1537.7(M+H)⁺ and the corresponding doubly charged ion at m/z 769.5 [½(M+2H)]⁺for the unsaturated carbocyclic peptide 114 and no starting linearpeptide 112.

Similar reaction conditions also resulted in complete cyclisation of113a to the isomeric [3,12]-dicarba analogue 115 (Scheme 6.17). Althougha higher catalyst loading (20 mol %) was required, the reaction went tocompletion in one hour. The enhancement in RCM yield via thismicrowave-assisted approach is remarkable in light of the poor resultsobtained using conventional heating methods. It is considered that theresults must be attributed to something beyond just more efficientheating. It has been postulated that another possible factor is thatmicrowave radiation causes highly efficient disruption of peptideaggregation on the solid support. It is noted that the reaction ofscheme 6.17 does not proceed without microwave irradiation.

On-resin Fmoc-deprotection of the unsaturated carbocycles 114a and 115afollowed by acid-mediated cleavage yielded the fully deprotectedpeptides 116 and 117. Aerial oxidation of 116 and 117 in 5%dimethylsulfoxide/aqueous ammonium carbonate (0.1 M, pH 8) then affordedthe unsaturated cystino-dicarba Ctx ImI analogues 118 and 119respectively (Scheme 6.18, Scheme 6.19). Each peptide was purified byreverse-phase HPLC (>99% purity) and isolated in 5% yield. Thesedicarba-analogues, and others based on native conotoxin sequences ofpharmaceutical significance (e.g. dicarba-analogues of conotoxinsextracted from Conus regius and Conus victoriae (ACV1)), seeexperimental section) are predicted to be biologically active due totheir similarities to the disulfide, and are predicted to have in vivostability due to the presence of the dicarba-bond.

It is important to note that the isolation and purification of nativeconotoxin sequences from cone snail venom is a low yielding and tediousprocess.²⁴⁷ Recently, 200 mg of venom extract from five cone snails(Conus textile) was purified to yield 1.1 mg (560 nmol) of conotoxinε-TxIX.²⁴⁸ Most references detailing the isolation of conotoxinmolecules from venom, however, do not cite isolation yields. Synthesisof conotoxin molecules can also be low yielding where oxidative foldingleads to several topoisomers.^(225,231,233,249-251) Extensivechromatography must be employed to isolate pure samples of the targetpeptide. Although the final purified yields of our dicarba-cystinoconotoxin analogues 118 and 119 were low, separation conditions were notoptimised and the scale of the reactions could be easily increased toafford larger quantities of pure peptide.

Hydrogenation of resin-bound unsaturated carbocyclic peptides 114a and115a was performed with Wilkinson's catalyst. This homogeneous catalystis ideal for this transformation as it allows reduction to be performedon the resin, operates under mild reaction conditions and is highlytolerant of sulfur-containing functionality. Hence, rhodium-catalysedhydrogenation of resin-bound carbocycles 114a and 115a indichloromethane:methanol (9:1) effected quantitative reduction of theolefin at room temperature and low hydrogen pressure (80 psi) (Scheme6.20). Interestingly, the crude product from each of these reactions wasobtained as a mixture of the cystine reduced (120, 121) and oxidised(122, 123) forms. It is important to note that the final targets 122 and123 are isomeric with the unsaturated deprotected precursor peptides,116 and 117 respectively. An analogous hydrogenation experiment spikedwith linear diallyl conotoxin sequence 112, the precursor to theunsaturated carbocycle 114, showed a molecular ion consistent with theformation of the dipropyl sidechain-containing peptide 124.²³⁵ This massspectral data strongly suggests that the catalyst is not poisoned by thetrityl-protected cysteine residues and that the hydrogenation conditionsneeded for olefin reduction are uncompromised. Hence, the speciescontributing to the peak at m/z 769.5 [½(M+2H)]⁺ are likely to be thefinal isomeric cystino-dicarba Ctx ImI peptides 122 and 123.

Further support for this hypothesis comes from the LC-MS traces of theproduct mixtures. In each case, a signal at t_(R)=6.01 min (122) andt_(R)=7.02 min (123) was observed. In comparison, the retention timesfor the isomeric unsaturated carbocycles 116 and 117, under identicalchromatographic conditions, are 5.66 min and 6.64 min respectively. Thesaturated cystino-dicarba α-conotoxin analogues 122 and 123 arecurrently undergoing chromatographic purification and are being assessedfor biological activity and in vivo stability. NMR spectroscopy willalso be used to further confirm the structures of the isomeric conotoxinanalogues 122 and 123.

6.4.2 Bis-Dicarba Conotoxin Analogues

The regioselective on-resin methodology described in section 4 was alsoapplied to the synthesis of fully carbocyclic conotoxin ImI analogues.The catalytic sequence involves the selective RCM of reactiveallylglycine units in the presence of dormant prenylglycine residuesfollowed by selective hydrogenation of the resultant unsaturatedcarbocycle. Activation of the prenyl groups via butenolysis gives theactive crotyl sidechains which can undergo the RCM-hydrogenation processto afford the target bicycles 125 and 126 (Scheme 6.21).

This study commenced with the construction of the linear isomericconotoxin analogues, [2,8]-Hag-[3,12]-Pre conotoxin ImI 127 and[2,8]-Pre-[3,12]-Hag conotoxin ImI 128. Standard SPPS techniquesemploying Rink amide resin, HATU-NMM activation and Fmoc-protected aminoacids facilitated synthesis of the peptides 127 and 128. Both of thesesequences possess two strategically placed non-proteinaceousL-allylglycine (Hag) residues to facilitate the selective constructionof the first carbocycle. The incorporation of two less reactiveprenylglycine residues later enables the selective formation of thesecond carbocycle. During construction of the linear peptides,intermediates were carried through without purification orcharacterisation up to the dodecapeptides 127 and 128. As expected, theprenylglycine residues were incorporated without complication and massspectral analysis gave doubly charged molecular ion peaks at m/z 805.6[½(M+2H)]⁺ and 816.6 [½(M+Na+H)]⁺ which are consistent with thestructures of the isomeric sidechain deprotected linear peptides 127 and128. An additional peak at m/z 814.6 [½(M+H₂O+2H)]⁺, corresponding tothe acid-promoted hydration of a prenyl group, was also apparent in thespectrum.

After confirming the successful synthesis of the linear peptides 127 and128, ring closing metathesis of the resin-tethered peptides wasperformed using conventional heating methods. Exposure of peptide 127ato second generation Grubbs' catalyst (40 mol %) in dichloromethane and10% lithium chloride in dimethylformamide at 50° C. for 40 hours gavethe unsaturated carbocycle 129 (Scheme 6.22).

Analogous RCM conditions for 128a, however, led to complete recovery ofthe linear peptide. These results highlight the influence of the peptidesequence on RCM success when microwave is not used. A derivative of theproblematic sequence 128 was therefore constructed to elucidate theeffect of a turn-inducer. A new peptide sequence 130 was synthesisedpossessing a Pro9 residue rather than the native Ala9 residue.Interestingly, the resultant solid-supported peptide 130a cyclised underthe previously unsuccessful metathesis conditions (without microwaveradiation) to give 131, but the RCM did not go to completion (Scheme6.23). Unfortunately, LC-MS analysis did not enable separation of thelinear 130 and cyclic 131 peptide and hence an estimation of reactionconversion could not be made.

Microwave-assisted ring closing metathesis, however, provided expedientsyntheses for both the target carbocycles 129 and 132. Microwaveirradiation of a solution of Rink amide bound-peptide 127a and secondgeneration Grubbs' catalyst (10 mol %) in dichloromethane containing 10%lithium chloride in dimethylformamide at 100° C. resulted in completering closure in only one hour (Scheme 6.24). Mass spectral analysis ofthe product mixture showed the required molecular ion with m/z 791.4[½(M+2H)]⁺ for the unsaturated dicarba peptide 129 and no startinglinear peptide 127.

The resin-bound isomeric dicarba analogue 128a also completely cyclisedin one hour with 20 mol % second generation Grubbs' catalyst using thesame solvent system at 100° C. (Scheme 6.25).

These results were very exciting and demonstrated the power of microwaveenergy to yield carbocycles that were unattainable by conventionalheating methods. In addition, the prenyl sidechains remained inert tothe microwave-accelerated metathesis conditions and no cross metathesisproducts were observed.

Rhodium-catalysed hydrogenation of the resin-bound carbocycles 129a and132a in dichloromethane:methanol (9:1) effected quantitative reductionof the unsaturated carbocycle at room temperature and low hydrogenpressure (80 psi) (Scheme 6.26 and Scheme 6.27). The mass spectra ofcleaved peptides from both reactions displayed doubly charged molecularion peaks at m/z 792.5 [½(M+2H)]⁺ and m/z 801.5 [½(M+H₂O+2H)]⁺confirming formation of the isomeric products 133 and 134. Importantly,the prenyl groups resisted hydrogenation and were now available foractivation to facilitate construction of the second carbocycle.

Activation of the prenyl sidechains involved butenolysis of thesolid-supported peptides 133a and 134a. The peptide 133a was exposed toan atmosphere of cis-2-butene (15 psi) and a mixture of 40 mol % secondgeneration Grubbs' catalyst and benzoquinone in dichloromethane for 38hours (Scheme 6.28). Benzoquinone was added to the reaction mixture toreduce or eliminate the potential for olefin isomerisation. Massspectral analysis of a cleaved aliquot of peptide confirmed formation ofthe target dicrotylglycine-containing peptide 135 with the appearance ofa peak at m/z 778.4 [½(M+2H)]⁺. No starting prenyl-containing peptide133 was observed, however, mass spectral data revealed low intensity,doubly charged higher homologue species separated by m/z+7 units. Underthe above described metathesis conditions, the generated crotylsidechain can isomerise to a terminal butenyl chain and then undergosecondary cross metathesis with cis-2-butene (Scheme 6.29). The productsarising from this process of isomerisation-cross metathesis areconsistent with the observed mass spectral data.

Reaction conditions were modified to minimise this competingisomerisation reaction. These changes involved the addition ofchaotropic salts and variation of catalyst loading and reaction time.This aim was realised, although to a small extend this was stillaccompanied by partially metathesised peptide 136 and starting material133.

Microwave-accelerated ring closing metathesis of the resin-tetheredpeptide 135a using second generation Grubbs' catalyst (20 mol %) indichloromethane and 10% lithium chloride in dimethylformamide affordedthe target peptide 140 in only one hour (Scheme 6.30). Preliminary LC-MSanalysis was encouraging with the appearance of the required doublycharged molecular ion peak at m/z 750.4 [(M+2H)]⁺, corresponding to thebicyclic peptide 140. Interestingly, a very low intensity peak at m/z764.4 was also evident which corresponded to the cyclic product of acontaminating isomerisation-butenolysis adduct. The Fmoc-deprotectedproduct 125 is being purified and submitted for biological testing.

Rhodium-catalysed hydrogenation of the resin-bound bicycle 140a wasperformed in dichloromethane:methanol (9:1) at room temperature underlow hydrogen pressure (80 psi) (Scheme 6.31). Preliminary mass spectraland LC-MS data of the isolated residue confirm the formation of thesaturated bicycle 126.

The problem of isomerization experienced during activation of thediprenyl-conotoxin sequence 133a (see FIG. 6.5) was subsequentlyreinvestigated. It was postulated that the highly non-polar ethylene and2-butene, used for activation of the prenyl groups, could beincompatible with the polystyrene resin support. Resins, such as Wangand Rink amide, swell well in polar solvents; exposure to non polarsolvents results in resin shrinkage, poor accessibility of reagents (iecatalysts) to reactive functionality and consequently poor conversion. Amore polar derivative of ethylene and 2-butene, however, would achievebetter resin swell and higher activation yields. Conseqeuntly, aninvestigation using 1,4-diacetoxy-cis-2-butene was initiated. Thismolecule has the advantage of being a liquid at ambient temperaturewhile exhibiting greater polarity than either ethylene or butene.

Before 1,4-diacetoxy-cis-2-butene, or other like analogues (such as1,4-dichloro-2-butene), could be used to activate prenyl-containingresin-tethered peptides, (i) its reactivity and ability to activatehindered olefins; (ii) compatibility with resin-tethered substrates; and(iii) its ability to form a reactive intermediate (i.e. allylic acetate)capable of a subsequent CM with a reactive type I olefin (i.e. to formsubsequent intra/inter dicarba bonds) needed to be investigated. Steps(i) and (iii) were investigated using a simple small molecule derivedfrom commercially available racemic allylglycine in three steps (Scheme6.32).

To generate the required type III olefin, the benzoyl-protected methylester of allylglycine 62 underwent a cross metathesis reaction with2-methyl-2-butene (Scheme 6.32). The reaction proceeded at 50° C. indichloromethane for 72 h in a pressurised vessel giving theprenylglycine derivative 87 in quantitative conversion afterchromatographic purification.

Next, the fully protected prenylglycine analogue 87 was subjected tostandard cross metathesis conditions using an excess of1,4-diacetoxy-cis-2-butene, in the presence of second generation Grubbs'catalyst (Scheme 6.32). After stirring at 50° C. overnight, the reactionwas complete, as indicated by t.l.c. Column chromatography of the crudematerial yielded the activated molecule 141 in 57% yield.

With activation of the prenylglycine derivative complete, the reactivityof the resultant molecule was assessed. Initially, homodimerisation wasattempted; the molecule was subjected to standard cross metathesisconditions, in the presence of second generation Grubbs' catalyst at 50°C. (Scheme 6.33). Mass spectral analysis indicated both the desiredproduct and starting material. Gas chromatographic analysis indicatedthe expected equilibrium statistical mixture of the desired homodimer69, 1,4-diacetoxy-2-butene and starting material 141.

To assess the reactivity of the activated moiety 141, cross metathesiswith a type I olefin was investigated (Scheme 6.31). Standard crossmetathesis conditions were applied, with a 6-fold excess of the type Iolefin to avoid the statistical distribution of products and increasethe yield of the desired peptide. The desired cross metathesis product142 was obtained as a brown oil in 81% yield following purification viacolumn chromatography. Spectroscopic analysis confirmed the presence ofboth the E- and Z-isomers, though individual NMR signals could not beassigned to a specific geometry.

To assess the compatibility of 1,4-diacetoxy-cis-2-butene withresin-tethered substrates, a simple prenylglycine-containing dipeptidewas subjected to a cross metathesis reaction with1,4-diacetoxy-cis-2-butene (Scheme 6.34). This reaction showed completeconversion of the resin-tethered type II olefin to the correspondingtype I olefin, indicating complete compatibility of1,4-diacetoxy-cis-2-butene with resin-bound substrates. No isomerizationof the double bond was observed leading to the conclusion that theextended reaction times needed during activation reactions usingnon-polar olefins is responsible for the competing isomerisationpathway.

6.5 Stability

Despite the known activity of conotoxins as therapeutics, their multipledisulfide bond frameworks are known to be unstable under reducingconditions. Reduction or framework scrambling by thiol containingmolecules such as glutathione or serum albumin in intracellular orextracellular environments such as blood plasma can decrease theireffectiveness as drugs.

Incubation of native-Ctx ImI in human blood plasma has been shown toproduce significant rearrangement of the disulfide framework (i.e.scrambling). Similarly, treatment of Ctx-IMI with glutathione, areducing enzyme commonly found in blood plasma, results in completescrambling of the disulfide framework in ˜6 hours. See for instanceArmishaw, C. J., Daly, N. L., Nevin, S. T., Adams, D. J., Craik, D. J.,Alewood, P. F., J. Biol. Chem., 2006, in press. Such scrambling orreduction is not possible with dicarba-Ctx IMI analogues; the dicarbalinkage is completely inert to such reductants.

6.6 Summary

In conclusion, the strategy developed can be used for the regioselectiveconstruction of multi-dicarba bond-containing peptides. The methodologywas successfully applied to a model synthetic pentapeptide 102 and ledto the regioselective construction of an intramolecular andintermolecular dicarba bridge. Similarly, a tandemmetathesis-hydrogenation sequence provided dicarba-cystino analogues ofnaturally occurring conotoxin ImI 93. Here, a microwave accelerated ringclosing metathesis provided cyclic peptides that were unattainable viaconventional heating methods. A fully carbocyclic analogue of conotoxinImI 140 was also synthesised. Although activation of the prenylglycineunits with non-polar olefins (such as 2-butene and ethene) wassignificantly retarded on the resin-bound peptide 133a, butenolysis didlead to the desired activated crotyl sidechains. The selectivity of themethodology was maintained when investigated in the heterogeneoussystem. An intramolecular dicarba bridge was selectively constructedfrom allylglycine units in the presence of two prenyl olefins. Amicrowave-promoted RCM of the resin-bound crotyl-containing peptideultimately afforded the desired bicycle 140, and reduction lead toconotoxin 126. The use on polar 2-butene analogues, such as1,4-diacteoxy-2-butene, was found to be more compatible with polystyrenesupports and led to improved resin swelling and activation yields. Thiswas illustrated in both solution phase model studies and onsolid-supported peptide substrates.

7.0 EXAMPLES 7.1 Instrumentation

Microwave reactions were carried out on a Personal Chemistry (nowBiotage) Smith Synthesiser. The instrument produces a continuousfocussed beam of microwave irradiation at 2.45 GHz with a maximum powerdelivery of 300 W, which reaches and maintains a selected temperature(100° C.). Reactions were performed in high pressure quartz microwavevessels fitted with self-sealing Teflon septa as a pressure reliefdevice, that were crimped in place. The vessels contained magneticstirrer beads and the pressure and temperature of each reaction wasmonitored continuously with an in built pressure transducer (located inthe lid) and infrared pyrometer respectively. Reaction times weremeasured from the time the microwave began heating until the reactionperiod had elapsed (cooling periods were not inclusive).

7.2 Intentionally Left Blank 7.3 Peptide Procedures 7.3.1 Materials andReagents

Peptides were synthesised in polypropylene Terumo syringes (10 mL)fitted with a polyethylene porous (20 μm) filter. Solid phase peptidesynthesis (SPPS) was performed using a Visprep™ SPE DL 24-port modelvacuum manifold supplied by Supelco. Coupling reactions and cleavagemixtures were shaken on a KS 125 basic KA elliptical shaker supplied byLabortechnik at 400 motions per minute. Cleaved peptides werecentrifuged on a HermLe Z200A centrifuge supplied by Medos at a speed of4500 cycles per minute.

N,N′-Dimethylformamide (DMF) was supplied by Auspep and stored over 4 Åmolecular sieves. Dichloromethane (DCM) was supplied by BDH and storedover 4 Å molecular sieves. Wang resin, Rink amide resin, piperidine andtrifluoroacetic acid (TFA) were used as supplied by Auspep. Phenol wasused as supplied by BDH. Diisopropylcarbodiimide (DIC),diisopropylethylamine (DIPEA), 4-(N,N′-dimethylamino)pyridine (DMAP),ethanedithiol (EDT), Ellman's reagent (5,5′-dithiobis(2-nitrobenzoicacid), N-methylmorpholine (NMM), and thioanisole were used as suppliedby Aldrich. (2S)-2-Aminopent-4-enoic acid (L-allylglycine, Hag) was usedas supplied by Peptech. N-Fluorenylmethoxycarbonylaminosuccinimide(Fmoc-OSu), O-(7-azabenzo-triazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluoro-phosphate (HATU) and sidechain-protected Fmoc-amino acidswere used as supplied by GL Biochem unless otherwise specified.

7.3.2 Peptide Synthesis Procedure

Peptides were prepared using general Fmoc-SPPS methodology.^(142,221)Manual SPPS was carried out using fritted plastic syringes, allowingfiltration of solution without the loss of resin. The tap fittedsyringes were attached to a vacuum tank and all washings were removed invacuo. This involved washing (or swelling) the resin in the requiredsolvent for a reported period of time, followed by evacuation whichallowed the removal of excess reagents before subsequent couplingreactions.

7.3.2.1 Wang Resin

In a fitted syringe, Wang resin was swollen with DCM (7 mL, 3×1 min,1×60 min) and DMF (7 mL, 3×1 min, 1×30 min). DIC (3 equiv.) was added toa solution of protected amino acid, Fmoc-L-Xaa-OH, (3 equiv.) in DMF (3mL). The activated amino acid solution was added to the swelled resinand shaken gently for 1 min. A solution of DMAP (0.3 equiv.) in DMF (1mL) was added to the resin and the reaction mixture was shaken gentlyfor the reported period of time. The mixture was then filtered and theresin-tethered amino acid was washed with DMF (7 mL, 3×1 min) to ensureexcess reagents were removed. In order to prevent formation of deletionproducts, remaining resin active sites were capped with an anhydridesolution (5% acetic anhydride, 1% NMM, 94% DMF) for 1 h. The mixture wasfiltered and the resin was washed with DMF (7 mL, 3×1 min) anddeprotected with 20% piperidine in DMF (7 mL, 1×1 min, 2×10 min). Afterthis deprotection step, the resin was washed with DMF (7 mL, 5×1 min) toremove traces of base prior to coupling the next amino acid.

Subsequent amino acids were coupled using the following procedure:

NMM (6 equiv.) was added to a solution of protected amino acid,Fmoc-L-Xaa-OH (3 equiv.) and HATU (2 equiv.) in DMF (3 mL) and shakengently for 1 min. The activated amino acid solution was added to theresin-tethered amino acid and shaken gently for the reported period oftime. The peptidyl-resin was then washed with DMF (7 mL, 3×1 min) andthe Kaiser test²⁵⁵ was performed to monitor coupling success. Anyincomplete reactions were repeated with extended reaction times(indicated in brackets). Upon negative test results for the presence offree amine, the resin-peptide was deprotected with 20% piperidine in DMF(7 mL, 1×1 min, 2×10 min) and washed again with DMF (7 mL, 5×1 min) toremove traces of base prior to coupling the next amino acid.

The above procedure was repeated until the desired peptide sequence wasconstructed. Once complete, the resin was washed with DMF (7 mL, 3×1min), DCM (7 mL, 3×1 min), MeOH (7 mL, 3×1 min), DCM (7 mL, 3×1 min),MeOH (7 mL, 3×1 min) and dried in vacuo for 1 h. A small aliquot ofresin-tethered peptide was then exposed to a TFA cleavage solution(Section 7.3.3).

7.3.2.2 Rink Amide Resin

In a fitted syringe, Rink amide resin was swollen with DCM (7 mL, 3×1min, 1×60 min) and DMF (7 mL, 3×1 min, 1×30 min) and deprotected with20% piperidine in DMF (7 mL, 1×1 min, 2×10 min) and washed again withDMF (7 mL, 5×1 min). NMM (6 equiv.) was added to a solution of aprotected amino acid, Fmoc-L-Xaa-OH (3 equiv.) and HATU (2 equiv.) inDMF (3 mL) and shaken gently for 1 min. The activated amino acidsolution was added to the resin and shaken gently for the reportedperiod of time. The peptidyl-resin was washed with DMF (7 mL, 3×1 min)to ensure excess reagents were removed. Kaiser tests were performed tomonitor coupling success and any incomplete reactions were repeated withextended reaction times (indicated in brackets). Upon negative testresults for the presence of free amine, the resin-peptide wasdeprotected with 20% piperidine in DMF (7 mL, 1×1 min, 2×10 min) andwashed again with DMF (7 mL, 5×1 min). This coupling procedure wasrepeated until the desired peptide sequence was constructed.

The above procedure was repeated until the desired peptide sequence wasconstructed. Once complete, the resin was washed with DMF (7 mL, 3×1min), DCM (7 mL, 3×1 min), MeOH (7 mL, 3×1 min), DCM (7 mL, 3×1 min),MeOH (7 mL, 3×1 min) and dried in vacuo for 1 h. A small aliquot ofresin-tethered peptide was then exposed to a TFA cleavage solution(Section 7.3.3).

Kaiser Test

The Kaiser test was performed in order to monitor coupling success bydetecting the presence of resin-bound free amines.^(221,255) Two dropsof 5% ninhydrin in EtOH, 80% phenol in EtOH and 2% v/v 0.001 M potassiumcyanide in pyridine were added to pre-washed (EtOH) resin beads in atube and the mixture was subsequently heated at 120° C. for 3-5 min.Blue colouration of the beads indicate the presence of free amines andprovide evidence for an incomplete coupling reaction. It should be notedthat this test cannot be performed after coupling asparagine, asparticacid, serine and proline.^(221,256)

7.3.3 Peptide Cleavage: TFA-Mediated Cleavage Procedure

A small aliquot of the resin-peptide (˜1 mg) was suspended in a cleavagesolution 2 mL):90% TFA:5% thioanisole:2.5% EDT 2.5% water and phenol(1.6 g/5 mL of cleavage solution) and shaken gently for 1.5 h. Themixture was then filtered and the resin beads were rinsed with TFA(2×0.5 mL). The filtrate was concentrated with a constant stream of airto yield an oil. The peptide was precipitated with ice-cold Et₂O (2 mL)and collected by centrifugation (3×10 min). The supernatant liquid wasdecanted and the resultant residue was collected and analysed by massspectrometry.

7.3.4 Ellman's Test

The Ellman's test was performed in order to monitor reaction progressduring thiol oxidation (cystine formation) by detecting the presence offree sulfhydryl groups.²⁵⁷ 200 of a solution of Ellman's reagent inaqueous (NH₄)₂CO₃ buffer (4 mg mL⁻¹ in 0.1 M buffer) was added to 200 μLof the reacting peptide solution. An intense yellow colouration of thesolution indicates the presence of free thiol groups and providesevidence for an incomplete oxidation reaction.

7.3.5 Automated Peptide Synthesis

Peptide synthesis was also performed on a CEM Liberty PeptideSynthesiser™ with a CEM Microwave Discover System™. Both systems wereoperated with the use of PepDriver software. The desired peptidesequence and methods were installed on PepDriver. The resin was weigheddirectly into a 50 mL centrifuge tube, DMF (5 mL) added, then the tubewas screwed into position on the Liberty resin manifold. Amino acidsolutions (0.2M in DMF) were prepared and installed onto the Libertyamino acid manifold. External reagents were prepared as described: A0.45M solution of HOBt and HBTU in DMF was prepared as the activatorreagent. A 20% v/v solution of piperidine in DMF was used at thedeactivation reagent. Activator base reagent was prepared by making a 2Msolution of DIEA in NMP. Delivery volumes of all external reagents werecalibrated on the Liberty Peptide Synthesizer™ prior to use. Thetemperature of the Discover System™ was maintained via a fiber opticsensor located below the microwave cavity.

For all automated synthesis, “B.01 Initial Deprotection” followed by“B.01 Extended Deprotection” cycles were used in the method. Thesedeprotection cycles consisted of washing with DMF (1×7 mL), addition ofthe deprotection reagent (20% piperidine in DMF, 10 mL), followed by the“B.01 Initial Deprotection” microwave program. The peptidyl-resin wasexposed to a temperature of 37° C. at a power of 37 watts for 2 min. Theresin was then washed with DMF (12 mL) and a further 10 mL of thedeprotection reagent was added followed by the “B.01 ExtendedDeprotection” cycle. The peptidyl-resin was exposed to a temperature of75° C. at a power of 45 watts for 10 min. The resin was then washed withDMF (3×7 mL). Amino acid coupling cycles varied for each type of peptideand these are specified in each automated peptide synthesis description.

7.4 Hydrogenation Procedures 7.4.1 Catalysts and Materials Catalysts:

Palladium on charcoal (Pd/C) with 10% Pd concentration was used assupplied by Aldrich and stored in a desiccator.Tris(triphenylphosphine)rhodium(I) chloride (Wilkinson's catalyst,Rh(I)(PPh₃)₃Cl]) was used as supplied by Aldrich and stored under argonin a dry box. Asymmetric catalysts:(+)-1,2-Bis[(2S,5S)-2,5-diethylphospholano]benzene(1,5-cyclooctadiene)rhodium(I)trifluoromethane-sulfonate ([(COD)Rh(I)—(S,S)-Et-DuPHOS]OTf,Rh(I)—(S,S)-Et-DuPHOS),(−)-1,2-bis[(2R,5R)-2,5-diethylphospholano]benzene(1,5-cyclooctadiene)rhodium(I)tetra-fluoroborate ([(COD)Rh(I)—(R,R)-Et-DuPHOS]BF₄,Rh(I)—(R,R)-Et-DuPHOS), (+)-1,2-bis[(2S,5S)-2,5-dimethylphospholano]benzene (1,5-cyclooctadiene)rhodium(I)trifluoromethanesulfonate ([(COD)Rh(I)—(S,S)-Me-DuPHOS] OTf,Rh(I)—(S,S)-Me-DuPHOS),(−)-1,2-bis[(2R,5R)-2,5-dimethylphospholano]benzene(1,5-cyclooctadiene)rhodium(I)tetrafluoroborate([(COD)Rh(I)—(R,R)-Me-DuPHOS]BF₄, Rh(I)—(R,R)-Me-DuPHOS), and(+)-1,2-bis[(2R,5R)-2,5-dimethylphospholano]ethane(1,5-cyclooctadiene)rhodium(I)trifluoromethanesulfonate ([(COD)Rh(I)—(R,R)-Me-BPE]OTf,Rh(I)—(R,R)-Me-BPE) andbis(carboxylato)[2,2′-bis(diphenylphosphino)-(R)-1,1-binapthyl]ruthenium(II)((S)—Ru—BINAP) were used as supplied by Strem Chemicals and stored underargon.

Gases:

Argon and hydrogen were supplied by BOC gases and were of high purity(<10 ppm oxygen). Additional purification was achieved by passage of thegases through water, oxygen and hydrocarbon traps.

Solvents:

Benzene, MeOH, DCM, ^(t)BuOH and THF used in metal-catalysedhydrogenation reactions were degassed with high purity argon prior touse.

Reaction Vessels:

Fischer-Porter shielded aerosol pressure reactors (100 mL) fitted withpressure gauge heads and stirrer beads were employed for hydrogenationreactions.

7.4.2 Pd/C Hydrogenation Procedure^(36,37)

A Fischer-Porter tube was charged with substrate, catalyst(substrate:catalyst, 50:1) and solvent (5-10 mL). The reaction vesselwas connected to the hydrogenation manifold, evacuated and flushed withargon gas before being charged with hydrogen gas to the reportedpressure. The reaction was stirred at the specified temperature for thereported period of time. The hydrogen gas was then vented, the catalystremoved via filtration through a Celite pad and the solvent evaporatedunder reduced pressure.

7.4.3 Asymmetric Hydrogenation Procedure^(36,37,119)

In a dry box, a Fischer-Porter tube was charged with substrate, catalyst(substrate:catalyst, 100:1) and dry deoxygenated solvent (4-10 mL). Thereaction vessel was assembled and tightly sealed within the dry box. Theapparatus was connected to the hydrogenation manifold and purged threetimes using a vacuum and argon flushing cycle before being pressurisedwith hydrogen gas to the reported pressure. The reaction was thenstirred at the specified temperature for the reported period of time.The hydrogen gas was vented and the solvent was evaporated under reducedpressure. Purification was achieved by flash chromatography (silica,EtOAc).

Freeze-Pump-Thaw Procedure

For liquid substrates, a freeze-pump-thaw cycle was applied and thesolution was transferred into a dry box and loaded into a Fischer-Portertube as described above. The substrate was dissolved in MeOH or benzenein a Teflon-sealed vessel. The solution was frozen upon immersion inliquid nitrogen and opened to a vacuum source (high vacuum line ˜0.05mm) to remove gases. The vessel was re-sealed and the solution wasallowed to thaw before being frozen with liquid nitrogen again. Thiscycle was repeated until gas evolution was no longer observed during thethaw cycle.

7.4.4 Wilkinson's Hydrogenation Procedure

In a dry box, a Fischer-Porter tube was charged with substrate,Wilkinson's catalyst (substrate:catalyst, 50:1) and dry deoxygenatedsolvent (4-10 mL). The apparatus was connected to the hydrogenationmanifold and purged three times using a vacuum and argon flushing cyclebefore being pressurised with hydrogen gas to the reported pressure. Thereaction was then stirred at ambient temperature for the reportedreaction time. The hydrogen gas was vented and the solvent wasevaporated under reduced pressure. Purification was achieved by flashchromatography (silica, EtOAc).

Hydrogenation experiments are described in the following format:substrate (mg), solvent (mL), catalyst, hydrogen pressure (psi),reaction temperature (° C.), reaction time (h), isolated yield (%),retention time (t_(R), GC/HPLC conditions) and enantiomeric excess(e.e.).

7.5 Metathesis Procedures 7.5.1 Catalysts and Materials Catalysts:

Bis(tricyclohexylphosphine)(benzylidene)ruthenium(II) dichloride(Grubbs' catalyst),tricyclohexylphosphine[1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene](benzylidene)ruthenium(II)dichloride (second generation Grubbs' catalyst) and1,3-bis(2,4,6-trimethylphenyl)-2-(imidazolidinylidene)dichloro-(o-iso-propoxyphenylmethylene)ruthenium(II)dichloride (second generation Hoveyda-Grubbs' second generationcatalyst) were used as supplied by Aldrich and stored under nitrogen.

Volatile Olefins:

Cis-2-butene (99%), cis+trans-2-butene (99%) 2-methylpropene(iso-butylene) and 2-methyl-2-butene were used as supplied by Aldrich.Ethylene was used as supplied by BOC gases.

Solvents:

DCM and a solution of lithium chloride in DMF (0.4 M LiCl/DMF) used inmetal-catalysed metathesis reactions were degassed with high purityargon prior to use.

Reaction Vessels:

Schlenk tubes and microwave reactor vessels fitted with stirrer beadswere employed for ring closing and cross metathesis reactions involvingthe use of solid or liquid (non-volatile) reactants. Fischer-Portershielded aerosol pressure reactors (100 mL) fitted with pressure gaugeheads and stirrer beads were employed for cross metathesis reactionsinvolving gaseous (ethylene, cis-2-butene, iso-butylene) or volatile(2-methyl-2-butene) reactants.

7.5.2 Conventional Ring Closing and Cross MetathesisProcedure^(116,142,152)

A Schlenk tube was charged with substrate(s), catalyst (5-40 mol %) anddeoxygenated solvent (˜5 mL) under an inert (nitrogen or argon)atmosphere. The reaction mixture was stirred at 50° C. for the specifiedperiod of time. Metathesis reactions were terminated upon exposure tooxygen and volatile species were removed under reduced pressure. Thecrude product was purified by flash chromatography.

7.5.3 Microwave-Accelerated Ring Closing and Cross Metathesis Procedure

A high pressure quartz microwave vessel was loaded with resin-tetheredpeptide, catalyst (5-40 mol %) and deoxygenated solvent (˜3-5 mL) underan inert (nitrogen and argon) atmosphere. The reaction mixture wasirradiated with microwaves and stirred at 100° C. for the reportedperiod of time. The mixtures were then filtered and washed with DMF (3mL, 3×1 min), DCM (3 mL, 3×1 min), MeOH (3 mL, 3×1 min) and dried on theSPPS manifold for 1 h. A small aliquot of resin-peptide (˜1 mg) was thensubjected to the TFA-mediated cleavage procedure (Section 7.3.3). Theisolated peptide was analysed by mass spectrometry.

Microwave-accelerated reactions were also performed on a CEM DiscoverSystem™. The instrument produces a continuous focused beam of microwaveirradiation at a power delivery of 40 W. The temperature on the DiscoverSystem™ was monitored via an infra-red sensor located below themicrowave cavity. Reactions were performed in a 10 mL high-pressurequartz vessel fitted with a self-sealing Teflon septa. The vessel wascharged with the peptidyl-resin, degassed solvent (5 mL DCM and 0.2 mL2M LiCl in DMF), 2^(nd) generation Grubb's catalyst (20 mol %) in aninert environment. The reaction mixture was irradiated with microwaveenergy whilst being stirred at 100° C. for 1 hr, cooled to roomtemperature, then terminated upon exposure to oxygen. The peptidyl-resinwas filtered through a fritted syringe and washed with DMF (5 mL, 3×1min), DCM (5 mL, 3×1 min), DMF (5 mL, 3×1 min) then MeOH (5 mL, 3×1 min)and dried in vacuo for 30 min prior to cleavage and analysis.

7.5.4 Conventional Cross Metathesis Procedure (Gaseous Reactant)

In a dry box, a Fischer-Porter tube was charged with substrate, catalyst(5-50 mol %) and deoxygenated solvent (˜5 mL). The reaction vessel wasthen evacuated and purged with ethylene, cis-2-butene or iso-butylene tothe reported pressure. The reaction mixture was stirred at 50° C. forthe specified period of time. Metathesis reactions were terminated uponexposure to oxygen and volatile species were removed under reducedpressure. The crude product was purified by flash chromatography.

7.5.5 Conventional Cross Metathesis Procedure (Volatile Reactant)

A Fischer-Porter tube was charged with substrate, catalyst (5 mol %),deoxygenated solvent (˜5 mL) and 2-methyl-2-butene. The reaction mixturewas stirred at 50° C. for the specified period of time. Metathesisreactions were terminated upon exposure to oxygen and volatile specieswere removed under reduced pressure. The crude product was purified byflash chromatography.

Metathesis experiments are described using the following format:substrate (mg), solvent (mL), catalyst (mg), reacting olefin (in thecase of cross metathesis) reaction temperature (° C.), reaction time(h), percent conversion (%). Chromatographic purification conditions(isolated yield, %) are also listed.

Hydrogenation and metathesis experiments performed on-resin weresubjected to the conditions described above. Resin-based metathesisreactions were quenched with ethyl vinyl ether (0.5 mL, 5 min). Themixtures were then filtered, washed with DCM (3 mL, 3×1 min), MeOH (3mL, 3×1 min) and dried on the SPPS manifold for 1 h. A small aliquot ofresin-peptide (˜1 mg) was subjected to the TFA-mediated cleavageprocedure (Section 7.33). The isolated peptide was analysed by massspectrometry.

Experimental for Section 4 7.6 Synthesis of 5,5-DimethylprolinePrecursors 7.6.1 N-Acetyl-2-hydroxyglycine 42

The titled compound 42 was prepared according to a procedure describedby Williams et al.¹⁹⁵ A solution of acetamide 34 (6.10 g, 0.10 mol) andglyoxylic acid monohydrate 41 (10.60 g, 0.14 mol) in anhydrous acetone(150 mL) was heated at reflux for 18 h. The reaction mixture wasevaporated under reduced pressure to afford the desired product 42 as aviscous yellow oil (13.75 g, 100%). Spectroscopic data indicated thecrude product 42 did not require purification and was used directly inthe subsequent reaction (Section 7.9.2). ν_(max) (neat): 3317bs, 2974w,1732s, 1668s, 1538s, 1379m, 1234w, 1112w, 1048m, 880m cm⁻¹. ¹H n.m.r.(300 MHz, D₆-DMSO): δ 1.84 (s, 3H, CH₃CO), 5.39 (d, J=8.7 Hz, 1H, H2),(8.65, bd, J=8.4 Hz, 1H, NH), two exchangeable protons (OH) notobserved. ¹³C n.m.r. (75 MHz, D₆-DMSO): δ 22.5 (CH₃CO), 70.9 (C2), 169.4(CONH), 171.5 (C1). Mass Spectrum (ESI⁺, MeOH): m/z 134.2 (M+H)⁺,C₄H₈NO₄ requires 134.1. Spectroscopic data were in agreement with thosereported in the literature.¹⁹⁵

7.6.2 Methyl N-Acetyl-2-methoxyglycinate 43

The methyl ester 43 was prepared according to a procedure described byLegall et al.¹⁹⁶ Concentrated H₂SO₄ (4.5 nit) was added to an ice-cooledsolution of N-acetyl-2-hydroxyglycine 42 (13.69 g, 0.10 mol) in MeOH(150 mL). The solution was stirred for 2 d at room temperature thenpoured into an ice-cooled saturated NaHCO₃ solution (400 mL). Themixture was extracted with EtOAc (3×250 mL) and the combined organicextract was dried (MgSO₄) and evaporated under reduced pressure to yieldthe titled compound 43 as a yellow oil (9.57 g, 60%). Spectroscopic dataindicated the crude product 43 did not require purification and was useddirectly in the subsequent reaction (Section 7.9.3). ν_(max) (neat):3334bm, 2955w, 1753s, 1671s, 1528m, 1439m, 1375m, 1221m, 1088m, 783wcm⁻¹. ¹H n.m.r. (300 MHz, CDCl₃): δ 2.10 (s, 3H, CH₃CO), 3.47 (s, 3H,OCH₃), 3.82 (s, 3H, COOCH₃), 5.55 (d, J=9.3 Hz, 1H, H2), 6.72 (bd, J=8.1Hz, 1H, NH). ¹³C n.m.r. (75 MHz, CDCl₃): δ 23.3 (CH₃CO), 53.0 (OCH₃),56.8 (COOCH₃), 78.3 (C2), 168.7 (CONH), 170.8 (C1). Mass Spectrum (ESI⁺,MeOH): m/z 184.1 (M+Na)⁺, C₆H₁₁NNaO₄ requires 184.2. Spectroscopic datawere in agreement with those reported in the literature.¹⁹⁶

7.6.3 Methyl 2-N-Acetylamino-2-(dimethoxyphosphinyl)acetate 39

The phosphinyl compound 39 was prepared according to a proceduredescribed by Schmidt et al.¹⁹⁷ Phosphorus (III) chloride (3.91 mL, 44.6mmol) was added to a solution of methyl N-acetyl-2-methoxyglycinate 43(7.19 g, 44.6 mmol) in toluene (100 mL) at 70° C. and the mixture wasstirred at this temperature for 17 h. Trimethyl phosphite (5.27 mL, 44.7mmol) was then added dropwise and the reaction mixture was left to stirfor 2 h at 70° C. The mixture was evaporated under reduced pressure andthe resultant oil was re-dissolved in DCM (100 mL) and washed withsaturated NaHCO₃ solution (3×100 mL). The organic extract was dried(MgSO₄) and evaporated under reduced pressure to afford the product 39as a colourless solid (1.46 g, 14%). The combined aqueous layers wereextracted in a continuous extractor with DCM (150 mL) for 3 d. Theorganic layer was then evaporated under reduced pressure to give theproduct 39 as a colourless solid (3.21 g, 30%) (44% combined yield),m.p. 89-91° C. (lit.¹⁹⁷ 88-89° C.). Spectroscopic data indicated thecrude product 39 did not require purification and was used directly inthe subsequent reaction (Section 7.94). ν_(max) (KBr): 3281m, 3050w,2852w, 1749m, 1673m, 1540m, 1309m, 1287w, 1232w, 1133m, 1061m, 1028mcm⁻¹. ¹H n.m.r. (300 MHz, CDCl₃): δ 2.08 (s, 3H, CH₃CO), 3.80-3.85 (m,9H, COOCH₃, 2×P—OCH₃), 5.23 (dd, J=22.2, 8.9 Hz, 1H, H2), 6.42 (d, J=8.8Hz, 1H, NH). ¹³C n.m.r. (75 MHz, CDCl₃): δ 22.7 (CH₃CO), 50.0 (d,J=146.8 Hz, C2), 53.3 (COOCH₃), 54.1 (d, J=6.8 Hz, P—OCH₃), 54.2 (d,J=6.5 Hz, P—OCH₃), 167.0 (d, J=2.2 Hz, CONH), 169.0 (d, J=6.0 Hz, C1).Mass Spectrum (ESI⁺, MeOH): m/z 262.1 (M+Na)⁺, C₆H₁₁NNaO₄ requires262.2.

7.7 Synthesis of Olefinic Substrates 7.7.1 (2Z)-Methyl2-N-Acetylaminopenta-2,4-dienoate 57

The dienamide 57 was prepared according to a procedure described by Teohet al.¹¹⁹ Tetramethylguanidine (3.22 mL, 25.7 mmol) and hydroquinone(10.0 mg) were added to a solution of methyl2-N-acetylamino-2-(dimethoxyphosphinyl)acetate 39 (4.63 g, 19.4 mmol) inTHF (60 mL) at −78° C. After 15 min, acrolein 58 (1.55 mL, 23.2 mmol)was added and the mixture was stirred at −78° C. for 2 h and then warmedto 25° C. and allowed to react an additional 2 h. The reaction mixturewas diluted with DCM (100 mL) and washed with dilute HCl solution (1 M,2×80 mL), CuSO₄ solution (1 M, 2×80 mL), saturated NaHCO₃ solution (2×80mL) and saturated NaCl solution (1×80 mL). The organic layer was dried(MgSO₄) and evaporated under reduced pressure to give the desireddienamide 57 as an off-white solid (2.78 g, 85%), m.p. 60-62° C.(lit.¹¹⁹ 61-63° C.). Spectroscopic data indicated the crude product 57did not require purification and was used directly in the subsequentreaction (Section 7.11.2). ν_(max) (KBr): 3277m, 3011m, 2955m, 1733s,1655s, 1594m, 1518s, 1438m, 1377w, 1350w, 1250w, 1113s, 1016m, 994m,950s, 768m cm⁻¹. ¹H n.m.r. (300 MHz, CDCl₃): δ 2.16 (s, 3H, CH₃CO), 3.81(s, 3H, OCH₃), 5.49 (d, J=9.9 Hz, 1H, H5-E), 5.61 (d, J=17.1 Hz, 1H,H5-Z), 6.47 (m, 1H, H4), 7.05 (d, J=11.1 Hz, 1H, H3), 7.07 (bs, 1H, NH).¹³C n.m.r. (75 MHz, CDCl₃): δ 23.6 (CH₃CO), 52.7 (OCH₃), 123.7 (C2),125.2 (C5), 132.0 (C4), 132.9 (C3), 165.5, 168.9 (C1, CONH). MassSpectrum (ESI⁺, MeOH): m/z 170.2 (M+1V, C₈H₁₂NO₃ requires 170.2.Spectroscopic data were in agreement with those reported in theliterature.¹¹⁹

7.7.2 (2S)-Methyl 2-N-Acetylaminopent-4-enoate 21a

The dienamide 57 was subjected to the general asymmetric hydrogenationprocedure (Section 7.4.3) under the following conditions: (2Z)-Methyl2-N-acetylaminopenta-2,4-dienoate 57 (108 mg, 0.64 mmol), benzene (7mL), Rh(I)—(S,S)-Et-DuPHOS, 30 psi, 22° C., 3 h. At the end of thereaction period, the solvent was evaporated under reduced pressure andthe residue was purified by flash chromatography (SiO₂, EtOAc) to give ayellow oil (106 mg, 97%). ¹H n.m.r. spectroscopy confirmed formation ofthe desired product 21a and the fully saturated compound, (2S)-methyl2-N-acetylaminopentanoate 59 (δ 0.93 (t, J=7.3 Hz, 3H, H5), 1.25-1.44(m, 4H, H3, 4)), in a 97:3 ratio respectively. GC: (2S)-21a t_(R)=18.6min (GC chiral column 50 CP2/XE60-SVALSAPEA, 100° C. for 1 min, 5° C.min⁻¹ to 280° C. for 9 min), 95% e.e. [α]_(D) ²²+45.0° (c=0.76, CHCl₃)containing 3% of 59 (lit.²⁰⁸ for (S)-21a [α]_(D) ²²+45.4° (c=3.57,CHCl₃)). ν_(max) (neat): 3278s, 3079w, 2955w, 1744s, 1657s, 1546m,1438m, 1375m, 1275w, 1226w, 1151m, 997w, 924w cm⁻¹. ¹H n.m.r. (300 MHz,CDCl₃): δ 2.00 (s, 3H, CH₃CO), 2.43-2.62 (m, 2H, H3), 3.73 (s, 3H,OCH₃), 4.67 (dt, J=11.6, 5.8 Hz, 1H, H2), 5.08 (m, 1H, H5-E), 5.14 (m,1H, H5-Z), 5.67 (m, 1H, H4), 6.06 (bs, 1H, NH). ¹³C n.m.r. (100 MHz,CDCl₃): δ 23.1 (CH₃CO), 36.5 (C3), 51.8 (C2), 52.4 (OCH₃), 119.2 (C5),132.3 (C4), 169.9, 172.4 (C1, CONH). Mass Spectrum (EST⁺, MeOH): m/z194.1 (M+Na)⁺, C₈H₁₃NNaO₃ requires 194.2. Spectroscopic data were inagreement with those reported in the literature.¹¹⁹

(2R)-Methyl 2-N-acetylaminopent-4-enoate 21a

The dienamide 57 was subjected to the general asymmetric hydrogenationprocedure (Section 7.4.3) under the following conditions: (2Z)-Methyl2-N-acetylaminopenta-2,4-dienoate 57 (40.0 mg, 0.24 mmol), benzene (5mL), Rh(I)—(R,R)-Et-DuPHOS, 30 psi, 22° C., 3 h. At the end of thereaction period, the solvent was evaporated under reduced pressure andthe residue was purified by flash chromatography (SiO₂, EtOAc) to giveas a yellow oil (36.0 mg, 88%). ¹H n.m.r. spectroscopy confirmedformation of the desired product 21a and the fully saturated compound,(2R)-methyl 2-N-acetylaminopentanoate 59 in a 95:5 ratio respectivelyGC: (2R)-21a t_(R)=18.2 min (GC chiral column 50 CP2/XE60-SVALSAPEA,100° C. for 1 min, 5° C. min⁻¹ to 280° C. for 9 min), 95% e.e. [α]_(D)²²−43.0° (c=0.47, CHCl₃) containing 5% of 59. Spectroscopic data were inagreement with those previously reported for the (S)-enantiomer.

7.7.3 N-Benzoyl-2-hydroxyglycine 65

The titled compound 65 was prepared according to a procedure describedby Zoller et al.²⁰⁹ A mixture of benzamide 35 (5.00 g, 41.3 mmol) andglyoxylic acid monohydrate 41 (4.32 g, 46.9 mmol) in anhydrous acetone(70 mL) was heated at reflux for 19 h. The reaction mixture wasevaporated under reduced pressure to afford the desired product 65 as acolourless solid (8.06 g, 100%), m.p. 198-200° C. (lit.²⁰⁹ 200-201° C.(dec)) Spectroscopic data indicated the crude product 65 did not requirepurification and was used directly in the subsequent reaction (Section7.11.4). ν_(max) (KBr): 3310bs, 3058w, 1728s, 1646s, 1602w, 1582w,1535s, 1491w, 1452w, 1315m, 1292w, 1254m, 1161m, 1097s, 1040m, 1002w,957m, 805w, 770w, 728m, 692m, 654m, 609w, 515m, 483w cm⁻¹. ¹H n.m.r.(300 MHz, D₆-DMSO): δ 5.60 (d, J=7.8 Hz, 1H, H2), 7.41-7.49 (m, 2H, H3′,5), 7.55 (m, 1H, H4′), 7.86-7.92 (m, 2H, H2′, 6′), 9.26 (d, J=7.8 Hz,1H, NH), two exchangeable OH protons not observed. ¹³C n.m.r. (75 MHz,D₆-DMSO): δ 71.7 (C2), 127.6, 128.3, 131.7 (Arom CH), 133.7 (C1′),166.0, 171.5 (C1, CONH). Mass Spectrum (ESI⁺, MeOH): m/z 218.2 (M+Na)⁺,C₉H₉NNaO₄ requires 218.2.

7.7.4 Methyl N-Benzoyl-2-methoxyglycinate 66

The methyl ester 66 was prepared according to a procedure described byZoller et al.²⁰⁹ Concentrated H₂SO₄ (2.0 mL) was added to an ice-cooledsolution of N-benzoyl-2-hydroxyglycine 65 (8.05 g, 41.3 mmol) in MeOH(65 mL). The solution was stirred for 48 h at ambient temperature thenpoured into an ice-cooled saturated NaHCO₃ solution (100 mL). Themixture was extracted with EtOAc (3×100 mL) and the combined organicextract was dried (MgSO₄) and evaporated under reduced pressure to yieldthe titled compound 66 as a yellow oil (8.00 g, 87%). Spectroscopic dataindicated the crude product 66 did not require purification and was useddirectly in the subsequent reaction (Section 7.11.5). ν_(max) (neat):3310bm, 2955m, 2837w, 1760s, 1651s, 1603w, 1580w, 1525s, 1488m, 1439m,1338w, 1286w, 1226w, 1198w, 1147w, 1108m, 1022w, 924m, 850w, 803m, 778m,692m cm⁻¹. ¹H n.m.r. (300 MHz, CDCl₃): δ 3.54 (s, 3H, OCH₃), 3.85 (s,3H, COOCH₃), 5.78 (d, J=9.0 Hz, 1H, H2), 7.22 (bd, J=9.0 Hz, 1H, NH),7.42-7.51 (m, 2H, H3′, 5), 7.56 (m, 1H, H4′), 7.80-7.88 (m, 2H, H2′,6′). ¹³C n.m.r. (75 MHz, CDCl₃): δ 53.2 (OCH₃), 57.0 (COOCH₃), 78.8(C2), 127.4, 128.9, 132.5 (Arom CH), 133.2 (C1′), 167.6, 168.7 (C1,CONH). Mass Spectrum (ESI⁺, MeOH): m/z 224.2 (M+H)⁺, C₁₁H₁₄NO₄ requires224.2; m/z 246.3 (M+Na)⁺, C₁₁H₁₃NNaO₄ requires 246.2. Spectroscopic datawere in agreement with those reported in the literature.²⁰⁹

7.7.5 Methyl 2-N-Benzoylamino-2-(dimethoxyphosphinyl)acetate 64

The phosphinyl compound 64 was prepared according to a proceduredescribed by Teoh et al.¹¹⁹ Phosphorus (III) chloride (3.15 mL, 36.0mmol) was added to a solution of methyl N-benzoyl-2-methoxyglycinate 66(8.00 g, 35.9 mmol) in toluene (70 mL) at 70° C. and the mixture wasstirred at this temperature for 14 h. Trimethyl phosphite (4.25 mL, 36.0mmol) was added dropwise and the reaction mixture was left to stir for 2h at 70° C. At the end of the reaction period, the mixture wasevaporated under reduced pressure and the resultant oil was re-dissolvedin DCM (100 mL) and washed with saturated NaHCO₃ solution (3×70 mL). Theorganic extract was isolated, dried (MgSO₄) and evaporated under reducedpressure to afford the titled compound 64 as a colourless solid (8.21 g,76%), m.p. 110-112° C. (lit.²¹⁰ 112-114° C.). Spectroscopic dataindicated the crude product 64 did not require purification and was useddirectly in the subsequent reaction (Section 7.11.6). ν_(max) (KBr):3300m, 3248m, 3059w, 2958m, 2852w, 1737s, 1671s, 1638m, 1618w, 1602w,1581w, 1541s, 1492m, 1432w, 1292s, 1235s, 1188w, 1152w, 1044s, 915m,881w, 832m, 812w, 791w, 780w, 758m, 708m, 616w, 562m cm⁻¹. ¹H n.m.r.(300 MHz, CDCl₃): δ 3.82-3.90 (m, 9H, COOCH₃, 2×P—OCH₃), 5.47 (dd,J=21.9, 9.0 Hz, 1H, H2), 6.97 (bd, J=7.8 Hz, 1H, NH), 7.43-7.49 (m, 2H,H3′, 5′), 7.56 (m, 1H, H4′), 7.82-7.87 (m, 2H, H2′, 6′). ¹³C n.m.r. (75MHz, CDCl₃): δ 50.6 (d, J=147.1 Hz, C2), 53.5 (COOCH₃), 54.2 (d, J=6.8Hz, P—OCH₃), 127.4, 128.8, 132.3 (Arom CH), 133.1 (C1′), 166.9 (d, J=5.4Hz, C1), 167.3 (d, J=2.0 Hz, CONH). Mass Spectrum (ESI⁺, MeOH): m/z302.2 (M+H)⁺, C₁₂H₁₇NO₆P requires 302.2; m/z 324.2 (M+Na)⁺, C₁₂H₁₆NNaO₆Prequires 324.2.

7.7.6 (2Z)-Methyl 2-N-Benzoylaminopenta-2,4-dienoate 63

The dienamide 63 was prepared according to a procedure described by Teohet al.¹¹⁹ Tetramethylguanidine (4.35 mL, 34.7 mmol) and hydroquinone(12.0 mg) were added to a solution of methyl2-N-benzoylamino-2-(dimethoxyphosphinyl)acetate 64 (7.79 g, 25.9 mmol)in THF (120 mL) at −78° C. After 30 min, acrolein 58 (2.10 mL, 31.4mmol) was added and the mixture was stirred at −78° C. for 2 h and thenwarmed to 25° C. and allowed to react an additional 2 h. The reactionmixture was diluted with DCM (150 mL) and washed with dilute HClsolution (1 M, 2×100 mL), CuSO₄ solution (1 M, 2×100 mL), saturatedNaHCO₃ solution (2×100 mL) and saturated NaCl solution (1×100 mL). Theorganic extract was dried (MgSO₄) and evaporated under reduced pressureto give the crude product 63 as a waxy-brown solid. Purification byflash chromatography (SiO₂, light petroleum:EtOAc:DCM, 3:2:2) furnishedthe pure enamide 63 as colourless needles (4.78 g, 80%), m.p. 138-141°C. (dec). (KBr): 3288bm, 2952m, 2361w, 1727s, 1652s, 1602w, 1580w,1514s, 1484s, 1436w, 1257s, 1196w, 1074w, 1028w, 991w, 931w, 800w, 737m,710m cm⁻¹. ¹H n.m.r. (300 MHz, CDCl₃): δ 3.83 (s, 3H, OCH₃), 5.50 (dd,J=10.0, 1.7 Hz, 1H, H5-K), 5.64 (dd, J=16.8, 1.7 Hz, 1H, H5-Z), 6.56(ddd, J=17.1, 11.4, 10.2 Hz, 1H, H4), 7.14 (d, J=11.2 Hz, 1H, H3),7.45-7.51 (m, 2H, H3′, 5′), 7.56 (m, 1H, H4′), 7.78 (bs, 1H, NH),7.88-7.91 (m, 2H, H2′, 6′). ¹³C n.m.r. (75 MHz, CDCl₃): δ 52.8 (OCH₃),123.6 (C2), 125.2 (C5), 127.6 (CT, 6′), 128.9 (C3′, 5′), 132.2, 132.2,132.3 (C3, 4, 4′), 133.9 (C1′), 165.6, 165.8 (C1, CONH). Mass Spectrum(ESI⁺, MeOH): m/z 232.1 (M+H)⁺, C₁₃H₁₄NO₃ requires 232.3; m/z 254.2(M+Na)⁺, C₁₃H₁₃NNaO₃ requires 254.2. Spectroscopic data were inagreement with those reported in the literature.²¹¹

7.7.7 (2S)-Methyl 2-N-Benzoylaminopent-4-enoate 62

The dienamide 63 was subjected to the general asymmetric hydrogenationprocedure (Section 7.4.3) under the following conditions: (2Z)-Methyl2-N-benzoylaminopenta-2,4-dienoate 63 (100 mg, 0.43 mmol), benzene (8mL), Rh(I)—(S,5)-Et-DuPHOS, 30 psi, 22° C., 3 h. At the end of thereaction period, the solvent was evaporated under reduced pressure andthe residue was purified by flash chromatography (SiO₂, EtOAc) to give apale yellow oil (100 mg, 99%). ¹H n.m.r. spectroscopy confirmedformation of the desired product 62 and the fully saturated compound,(2S)-methyl 2-N-benzoylaminopentanoate 68 (δ 0.95 (t, J=7.3 Hz, 3H, H5),1.36-1.50 (m, 2H, H4), 1.90-1.96 (m, 2H, H3)), in a 93:7 ratiorespectively. GC: (2S)-62 t_(R)=27.0 min (GC chiral column 50CP2/XE60-SVALSAPEA, 180° C. for 1 min, 2° C. min⁻¹ to 210° C. for 20min), 100% e.e. [α]_(D) ²²+49.3° (c=1.12, CHCl₃) containing 7% of 68.ν_(max) (neat): 3325bw, 3062w, 2955w, 2360w, 1743s, 1644s, 1603w, 1580w,1538m, 1489m, 1438w, 1360w, 1268w, 1225w, 1159w, 1075w, 1028w, 925m,802w, 714w, 668w cm⁻¹. ¹H n.m.r. (400 MHz, CDCl₃): δ 2.63-2.73 (m, 2H,H3), 3.79 (s, 3H, OCH₃), 4.91 (m, 1H, H2), 5.15 (m, 1H, H5-E), 5.18 (m,1H, H5-Z), 5.75 (m, 1H, H4), 6.67 (bd, J=7.0 Hz, 1H, NH), 7.42-7.46 (m,2H, H3′, 5′), 7.52 (m, 1H, H4′), 7.78-7.81 (m, 2H, H2′, 6′). ¹³C n.m.r.(100 MHz, CDCl₃): δ 36.8 (C3), 52.1 (C2), 52.6 (OCH₃), 119.5 (C5), 127.2(C2′, 6′), 128.8 (C3′, 5′), 131.9 (C4), 132.4 (C4′), 134.1 (C1′), 167.0,172.4 (C1, CONH). Mass Spectrum (ESI⁺, MeOH): m/z 234.3 (M+H)⁺,C₁₃H₁₆NO₃ requires 234.3; m/z 256.2 (M+Na)⁺, C₁₃H₁₅NNaO₃ requires 256.3.Spectroscopic data were in agreement with those reported in theliterature.²¹²

(2R)-Methyl 2-N-benzoylaminopent-4-enoate 62

The dienamide 63 was subjected to the general asymmetric hydrogenationprocedure (Section 7.4.3) under the following conditions: (2Z)-Methyl2-N-benzoylaminopenta-2,4-dienoate 63 (100 mg, 0.43 mmol), benzene (8mL), Rh(I)—(R,R)-Et-DuPHOS, 30 psi, 22° C., 3 h. At the end of thereaction period, the solvent was evaporated under reduced pressure andthe residue was purified by flash chromatography (SiO₂, EtOAc) to give ayellow oil (93.8 mg, 93%). ¹H n.m.r. spectroscopy confirmed formation ofthe desired product 62 and the fully saturated compound, (2R)-methyl2-N-benzoylaminopentanoate 68, in a 91:9 ratio respectively. GC: (2R)-62t_(R)=26.4 min (GC chiral column 50 CP2/XE60-SVALSAPEA, 180° C. for 1min, 2° C. min⁻¹ to 210° C. for 20 min), 100% e.e. [α]_(D) ²²−49.7°(c=0.64, CHCl₃) containing 9% of 68. Spectroscopic data were inagreement with those previously reported for the (S)-enantiomer.

7.7.8 (2Z)-Methyl 2-N-Acetylamino-5-phenylpenta-2,4-dienoate 76

The dienamide 76 was prepared according to a procedure described by Burket al.¹¹⁷ Tetramethylguanidine (0.70 mL, 5.58 mmol) was added to asolution of methyl 2-N-acetylamino-2-(dimethoxyphosphinyl)acetate 64(1.00 g, 4.18 mmol) in THF (50 mL) at −78° C. After 15 min,trans-cinnamaldehyde 78 (0.63 mL, 5.00 mmol) was added and the mixturewas stirred at −78° C. for 2 h, warmed to 25° C. and allowed to react anadditional 2 h. The reaction mixture was diluted with DCM (100 mL) andwashed with dilute HCl solution (1 M, 2×75 mL), CuSO₄ solution (1 M,2×75 mL), saturated NaHCO₃ solution (2×75 mL) and saturated NaClsolution (1×75 mL). The organic layer was dried (MgSO₄) and evaporatedunder reduced pressure to give the crude product 76 as a waxy solid(0.87 g). Purification by recrystallisation from a mixture of lightpetroleum, EtOAc and DCM furnished the pure dienamide 76 as an off-whitesolid (0.76 g, 74%), m.p. 180-181° C. (lit.¹¹⁷ 179-180° C.). ν_(max)(KBr): 3263w, 1721s, 1662s, 1517s, 1439m, 1368m, 1286m, 1229s, 1192w,1116m, 993m, 769w, 752m, 728w, 692m, 600w cm⁻¹. ¹H n.m.r. (300 MHz,CDCl₃): δ 2.20 (s, 3H, CH₃CO), 3.82 (s, 3H, OCH₃), 6.89-6.91 (m, 2H, H3,4), 7.01 (m, 1H, H5), 7.22 (bd, J obscured by residual CHCl₃ peak, 1H,NH), 7.29-7.37 (m, 3H, H3′, 4′, 5′), 7.45-7.48 (m, 2H, H2′, 6′). ¹³Cn.m.r. (100 MHz, CDCl₃): δ 23.9, (CH₃CO), 52.7 (OCH₃), 123.0 (C2),124.0, 127.5, 128.9, 129.2, 132.8, 140.2 (Arom CH, C3, 4, 5), 136.5(C1′), 165.6, 168.7 (C1, CONH). Mass Spectrum (ESI⁺, MeOH): m/z 246.2(M+H)⁺, C₁₄H₁₆NO₃ requires 246.3. Spectroscopic data were in agreementwith those reported in the literature.¹¹⁷

7.7.9 (2S)-Methyl 2-N-Acetylamino-5-phenylpent-4-enoate 77

The dienamide 76 was subjected to the general asymmetric hydrogenationprocedure (Section 7.4.3) under the following conditions: (2Z)-Methyl2-N-acetylaminopenta-2,4-dienoate 76 (28.0 mg, 0.11 mmol), MeOH (7 mL),Rh(I)—(S,5)-Et-DuPHOS, 90 psi, 22° C., 2 h. At the end of the reactionperiod, the solvent was evaporated under reduced pressure and theresidue was purified by flash chromatography (SiO₂, EtOAc) to give ayellow oil (27.2 mg, 96%). ¹H n.m.r. spectroscopy confirmed formation ofthe desired product 77 and the fully saturated compound, (2S)-methyl2-N-acetylamino-5-phenylpentanoate 79 (δ 1.60-1.87 (m, 4H, H3, 4),2.48-2.53 (m, 2H, H5), 3.72 (s, 3H, OCH₃), 4.65 (m, 1H, H2)), in a 91:9ratio respectively. [α]_(D) ²²+90.0° (c=0.64, CHCl₃) containing 9% of79. ν_(max) (neat): 3280bw, 3070m, 2960w, 2350w, 1745s, 1648s, 1605w,1575w, 1550m, 1478m, 1440w, 1369w, 1270w, 1225w, 1153w, 1075w, 1028w,925m, 805w, 720w cm⁻¹. ¹H n.m.r. (400 MHz, CDCl₃): δ 2.02 (s, 3H,CH₃CO), 2.64-2.78 (m, 2H, H3), 3.76 (s, 3H, OCH₃), 4.77 (m, 1H, H2),6.00-6.09 (m, 214, H4, NH), 6.45 (d, J=15.8 Hz, 1H, H5), 7.20-7.34 (m,5H, Arom CH). ¹³C n.m.r. (100 MHz, CDCl₃): δ 23.3 (CH₃CO), 36.0 (C3),52.1, 52.6 (C2, OCH₃), 123.6, 126.4, 127.8, 128.7, 134.3 (Arom CH, C4,5), 136.9 (C1′), 171.5, 172.5 (C1, CONH). Mass Spectrum (ESI⁺, MeOH):m/z 248.1 (M+H)⁺, C₁₄H₁₈NO₃ requires 248.2. Spectroscopic data were inagreement with those reported in the literature.¹¹⁷

(2R)-Methyl 2-N-Acetylamino-5-phenylpent-4-enoate 77

The dienamide 76 was subjected to the general asymmetric hydrogenationprocedure (Section 7.4.3) under the following conditions: (2Z)-Methyl2-N-acetylamino-5-phenylpenta-2,4-dienoate 76 (27.4 mg, 0.11 mmol), MeOH(5 mL), Rh(I)—(R,R)-Et-DuPHOS, 90 psi, 22° C., 2 h. At the end of thereaction period, the solvent was evaporated under reduced pressure andthe residue was purified by flash chromatography (SiO₂, EtOAc) to give ayellow oil (25.7 mg, 93%). ¹H n.m.r. spectroscopy confirmed formation ofthe desired product 77 and the fully saturated compound, (2R)-methyl2-N-acetylamino-5-phenylpentanoate 79 in a 87:13 ratio respectively.[α]_(D) ²²−89.8° (c=1.03, CHCl₃) containing 13% of 79. Spectroscopicdata were in agreement with those previously reported for the(S)-enantiomer.

7.7.10 (2Z)-Methyl 2-N-Acetylamino-5-methylhexa-2,4-dienoate 20

The preparation of (2Z)-methyl 2-N-acetylamino-5-methylhexa-2,4-dienoate20 from the phosophonate 39 has been previously described (Section7.9.4).

7.7.11 (2S)-Methyl 2-N-Acetylamino-5-methylhex-4-enoate 19

The preparation of (2S)-methyl 2-N-acetylamino-5-methylhex-4-enoate 19via asymmetric hydrogenation of dienoate 20 has been previouslydescribed (Section 7.9.5).

Metathesis Reactions with Olefinic Substrates 7.8.1 (2S,7S)-Dimethyl2,7-N,N′-Diacetylaminooet-4-enedioate 60

The dimer 60 was prepared via the conventional cross metathesisprocedure (Section 7.5.2) under the following conditions: (2S)-Methyl2-N-acetylaminopent-4-enoate 21a (95.0 mg, 0.56 mmol), DCM (4 mL),Grubbs' catalyst (91.0 mg, 0.11 mmol, 20 mol %), 50° C., 20 h, 100%conversion into 60. Purification by flash chromatography (SiO₂,DCM:light petroleum:EtOAc, 1:1:1→10% MeOH:DCM) furnished pure dimer 60as a brown oil (76.7 mg, 88%). GC: t_(R) (E/Z)=12.7 min, 12.8 min (GCcolumn 30QC5/BPX5, 150° C. for 1 min, 10° C. min⁻¹ to 280° C. for 6min). [α]_(D) ²²+92.0° (c=0.004, CHCl₃). ν_(max) (neat): 3286bm, 2956m,2931m, 2856w, 1742s, 1659s, 1542m, 1438m, 1375m, 1267m, 1220m, 1138w,1017w cm⁻¹. ¹H n.m.r. (300 MHz, CDCl₃): δ 2.04 (s, 6H, CH₃CO), 2.40-2.50(m, 4H, H3, 6), 3.74 (s, 6H, OCH₃), 4.64-4.70 (m, 2H, H2, 7), 5.36-5.40(m, 2H, H4, 5), 6.34 (bd, J=7.2 Hz, 2H, NH). ¹³C n.m.r. (100 MHz,CDCl₃): δ 23.1 (CH₃CO), 35.1 (C3, 6), 51.7 (C2, 7), 52.6 (OCH₃), 128.8(C4, 5), 170.3, 172.6 (C1, 8, CONH). HRMS (ESI⁺, MeOH). Found: m/z337.1375 (M+Na)⁺, C₁₄H₂₂N₂NaO₆ requires 337.1376. Spectroscopic datawere in agreement with those reported in the literature.²⁶⁴

7.8.2 (2S,7S)-Dimethyl 2,7-N,N′-Dibenzoylaminooct-4-enedioate 69

Method 1:

The dimer 69 was prepared via the conventional cross metathesisprocedure (Section 7.5.2) under the following conditions: (2S)-Methyl2-N-benzoylaminopent-4-enoate 62 (49.0 mg, 0.21 mmol), DCM (5 mL),Grubbs' catalyst (34.6 mg, 42.1 μmol, 20 mol %), 50° C., 18 h, 100%conversion into 69. Purification by flash chromatography (SiO₂,DCM:light petroleum:EtOAc, 1:1:1) gave pure dimer 69 as a pale brownsolid (37.8 mg, 82%), m.p. 140-142° C. GC: t_(R) (E/Z)=13.5, 13.9 min(GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min⁻¹ to 280° C. for 6min). [α]_(D) ²²+56.4°=0.27, CHCl₃). ν_(max) (KBr): 3322bm, 2953m,2358w, 1742s, 1644s, 1603w, 1580w, 1538m, 1488m, 1436m, 1267w, 1218m,1027w, 973w, 802w, 736m cm⁻¹. ¹H n.m.r. (300 MHz, CDCl₃): δ 2.57-2.69(m, 4H, H3, 6), 3.67 (s, 6H, OCH₃), 4.85-4.98 (m, 2H, H2, 7), 5.49 (t,J=4.1 Hz, 2H, H4, 5), 6.86 (bd, J=7.4 Hz, 2H, NH), 7.40-7.44 (m, 4H,H3′, 5′), 7.48-7.52 (m, 2H, H4′), 7.81-7.83 (m, 4H, H2′, 6′). ¹³C n.m.r.(100 MHz, CDCl₃): δ 35.2 (C3, 6), 52.5 (C2, 7), 52.6 (OCH₃), 127.2 (C2′,6′), 128.7 (C3′, 5′), 128.8 (C4, 5), 131.9 (C4′), 133.9 (C1′), 167.1,172.4 (C1, 8, CONH). HRMS (ESI⁺, MeOH). Found: m/z 461.1695 (M+Na)⁺,C₂₄H₂₆N₂NaO₆ requires 461.1689.

Method 2:

The dimer 69 was also prepared via the conventional cross metathesisprocedure (Section 7.5.2) under the following conditions: (2S)-Methyl2-N-benzoylaminopent-4-enoate 142, DCM (5 mL), Grubbs' catalyst (20 mol%), 50° C., 20 h, 100% conversion into 69.

7.8.3 Dimerisation of (2S)-Methyl 2-N-Benzoylaminopent-4-enoate 62 inthe presence of (2S,7S)-Dimethyl 2,7-N,N′-Diacetylaminooct-4-enedioate60

The olefinic mixture 62 and 60 was subjected to the conventional crossmetathesis procedure (Section 7.5.2) under the following conditions:

Method A:

(2S)-Methyl 2-N-benzoylaminopent-4-enoate 62 (37.0 mg, 0.16 mmol),(2S,7S)-dimethyl 2,7-N,N′-diacetylaminooct-4-enedioate 60 (29.5 mg, 93.9μmol), DCM (3 mL), 2^(nd) generation Grubbs' catalyst (13.5 mg, 15.9μmol, 10 mol %), 50° C., 15 h. Spectroscopic data indicated the presenceof the starting acetyl-allylglycine dimer 60, the benzoyl-allylglycinedimer 69 and additional peaks which mass spectrometry indicated could beattributed to the “mixed” cross metathesis product, (2S,7S)-dimethyl2-N-acetylamino-7-N-benzoylaminooct-4-enedioate 70. ¹H n.m.r.spectroscopic data for the homodimers 60 and 69 were in agreement withthose previously reported (Section 7.12.1 and Section 7.12.2). Theheterodimer 70 was detected by mass spectrometry. Mass spectrum (ESI⁺,MeOH): m/z 337.2 (M+Na)⁺ ₆₀, C₁₄H₂₂N₂NaO₆; m/z 399.3 (M+Na)⁺ ₇₀,C₁₉H₂₄N₂NaO₆; m/z 461.2 (M+Na)⁺ ₆₉, C₂₄H₂₆N₂NaO₆ requires 461.1689.

Method B:

(2S)-Methyl 2-N-benzoylaminopent-4-enoate 62 (37.0 mg, 0.16 mmol),(2S,7S)-dimethyl 2,7-N,N′-diacetylaminooct-4-enedioate 60 (30.0 mg, 95.5μmol), DCM (4 mL), Grubbs' catalyst (26.1 mg, 31.7 μmol, 20 mol %), 50°C., 18 h, 100% conversion of 62 into dimer 69. Dimer 60 was recoveredunchanged. Spectroscopic data for dimers 60 and 69 were in agreementwith those previously reported (Section 7.12.1 and Section 7.12.2). No“mixed” cross metathesis product, heterodimer 70, was observed.

7.8.4 Attempted Dimerisation of (2Z)-Methyl2-N-Acetylaminopenta-2,4-dienoate 57

The dienamide 57 was subjected to the conventional cross metathesisprocedure (Section 7.5.2) under the following conditions: (2Z)-Methyl2-N-acetylaminopenta-2,4-dienoate 57 (33.0 mg, 0.20 mmol), DCM (3 mL),Grubbs' catalyst (34.0 mg, 41.3 μmol, 20 mol %), 50° C., 15 h, 0%conversion into dimer 61. The dienamide 57 did not react under theseconditions. ¹H n.m.r. spectroscopic data for the recovered dienamide 57were in agreement with those previously reported (Section 7.11.1).

7.8.5 Attempted Dimerisation of (2S)-Methyl 2-N-Acetylaminopent-4-enoate21a in the presence of (2Z)-Methyl 2-N-Acetylaminopenta-2,4-dienoate 57

The mixture of olefins 21a and 57 was subjected to the conventionalcross metathesis procedure (Section 7.5.2) under the followingconditions: (2S)-Methyl 2-N-acetylaminopent-4-enoate 21a (34.0 mg, 0.20mmol), (2Z)-methyl 2-N-acetylaminopenta-2,4-dienoate 57 (33.6 mg, 0.20mmol), DCM (4 mL), Grubbs' catalyst (16.3 mg, 19.8 μmol, 10 mol %), 50°C., 18 h. The ¹H n.m.r. spectrum displayed peaks characteristic of thestarting allylglycine derivative 21a and dienamide 57 but no peakscharacteristic of expected dimer 60. The mass spectrum displayed peaksattributed to the allylglycine derivative 21a and thetricyclohexylphosphine-dienamide conjugate addition adduct, (2S)-methyl2-N-acetylamino-5-tricyclohexylphosphinylpent-2-enoate 143. MassSpectrum (ESI⁺, DCM/MeOH): m/z 194.1 (M+Na)⁺ _(21a), C₈H₁₃NNaO₃; m/z450.4 (M⁺)₁₄₃, C₂₆H₄₅NO₃P⁺.

7.8.6 Attempted Dimerisation of (2S)-Methyl2-N-Benzoylaminopent-4-enoate 62 in the presence of (2Z)-Methyl2-N-Acetylaminopenta-2,4-dienoate 57

The mixture of olefins 57 and 62 was subjected to the conventional crossmetathesis procedure (Section 7.5.2) under the following conditions:(2S)-Methyl 2-N-benzoylaminopent-4-enoate 62 (46.0 mg, 0.20 mmol),(2Z)-methyl 2-N-acetylaminopenta-2,4-dienoate 57 (33.4 mg, 0.20 mmol),DCM (4 mL), 2^(nd) generation Grubbs' catalyst (16.8 mg, 19.8 μmol, 10mol %), 50° C., 4.5 h. The reaction mixture was evaporated under reducedpressure to afford a dark brown oil (97.9 mg). The ¹H n.m.r. spectrumdisplayed peaks characteristic of the starting allylglycine derivative62, dienamide 57, traces of the target allylglycine dimer 69 andadditional peaks which were difficult to characterise. Mass spectrometrydisplayed peaks attributed to the allylglycine derivative 62, dienamide57, allylglycine dimer 69, dienamide dimer (2S,7S)-dimethyl2,7-N,N′-diacetylaminooct-2,4,6-trienedioate 61, “mixed”dienamide-allylglycine dimer (2S,7S)-dimethyl2-N-acetylamino-7-N-benzoyl-aminoocta-2,4-dienedioate 144 and thetricyclohexylphosphine-dienamide conjugate addition adduct 143.

Mass Spectrum (ESI⁺, DCM/MeOH): m/z 256.1 (M+Na)⁺ ₆₂, C₁₃H₁₅NNaO₃; m/z337.3 (M+Na)⁺ ₆₁, C₁₄H₁₈N₂NaO₆; m/z 397.3 (M+Na)⁺ ₁₄₄, C₁₉H₂₂N₂NaO₆; m/z450.4 (M)⁺ ₁₄₃, C₂₆H₄₅NO₃P⁺; m/z 461.3 (M+Na)⁺ ₆₉, C₂₄H₂₆N₂NaO₆ requires461.5.

7.8.7 NMR Study of Grubbs' Catalyst with Dienamide 57

In a dry box, a Teflon-sealed n.m.r. tube was charged with (2S)-methyl2-N-acetylaminopenta-2,4-dienoate 57 (10.8 mg, 63.9 μmol), Grubbs'catalyst (50.7 mg, 61.6 μmol) and degassed deuterated DCM (CD₂Cl₂, 0.8mL) at room temperature. The n.m.r. tube was shaken gently and reactionprogress was monitored by ¹H and ³¹P n.m.r. spectroscopy. Compounds wereidentified by the following diagnostic resonances: ¹H n.m.r. (300 MHz,CD₂Cl₂): After 15 min: Grubbs' catalyst: δ 8.61 (d, J=7.6 Hz, 2H,ortho-Arom CH), 20.05 (s, 1H, [Ru]═CHPh); Ruthenium-dienamide complex73: δ 7.96 (d, J=11.0 Hz, 1H, [Ru]═CH═CH), 20.11 (d, J=11.0 Hz, 1H,[Ru]═CH); Ruthenium-dienamide chelate 74 (trace): δ 15.20 (d, J=4.2 Hz,1H, [Ru]═CH); Ratio of ruthenium complexes [Ru]═CHPh:73:74=1.0:1.0:<0.1.After 60 min: Grubbs' catalyst: δ 8.45 (d, J=7.6 Hz, 2H, ortho-Arom CH),20.04 (s, 1H, [Ru]═CHPh); Ruthenium-dienamide complex 73: δ 7.96 (d,J=11.0 Hz, 1H, [Ru]═CH═CH), 20.10 (d, J=11.0 Hz, 1H, [Ru]═CH);Ruthenium-dienamide chelate 74: δ 6.73 (d, J=3.0 Hz, 1H, [Ru]═CH═CH),15.19 (d, J=4.2 Hz, 1H, [Ru]═CH); Ratio of ruthenium complexes[Ru]═CHPh:73:74=3:1:1. After 120 min (no change after 18 h):Ruthenium-dienamide chelate 74: δ 6.71 (d, J=3.0 Hz, 1H, [Ru]═CH═CH),15.19 (d, J=4.0 Hz, 1H, [Ru]═CH). ³¹P n.m.r. (300 MHz, CDCl₃): δRuthenium-dienamide chelate 74:35.0; Grubbs' catalyst: 37.0;Ruthenium-dienamide complex 73: 38.8; Tricyclohexylphosphine oxide:46.5.

7.8.8 NMR Study of Grubbs' Catalyst with Dienamide 76

In a dry box, a Teflon-sealed n.m.r. tube was charged with (2S)-methyl2-N-acetylamino-5-phenylpenta-2,4-dienoate 76 (10.0 mg, 40.8 μmol),Grubbs' catalyst (33.6 mg, 40.9 μmol) and degassed CD₂Cl₂ (0.8 mL) atroom temperature. The n.m.r. tube was shaken gently and reactionprogress was monitored by ¹H n.m.r. spectroscopy. After 4 h,ruthenium-vinylalkylidene formation was not observed and only peakscorresponding to Grubbs' catalyst and the starting dienamide 76 werepresent.

7.8.9 Dimerisation of (2S)-Methyl 2-N-Acetylamino-pent-4-enoate 21a inthe presence of (2Z)-Methyl 2-N-Acetylamino-5-phenylpenta-2,4-dienoate76

The mixture of olefins 21a and 76 was subjected to the conventionalcross metathesis procedure (Section 7.5.2) under the followingconditions: (2S)-Methyl 2-N-acetylaminopent-4-enoate 21a (18.1 mg, 0.11mmol), (2Z)-methyl 2-N-acetylamino-5-phenylpenta-2,4-dienoate 76 (26.1mg, 0.11 mmol), DCM (4.0 mL), Grubbs' catalyst (8.7 mg, 10.6 μmol, 10mol %), 50° C., 18 h, 28% conversion of allylglycine 21a into 60.Dienamide 76 did not react under these conditions. n.m.r. spectroscopicdata for dienamide 76, dimer 60 and recovered allylglycine derivative21a were in agreement with those previously reported (Section 7.11.8,Section 7.12.1 and Section 7.11.2 respectively).

7.8.10 Dimerisation of (2S)-Methyl 2-N-Acetylamino-5-phenylpent-4-enoate77

The enamide 77 was subjected to the conventional cross metathesisprocedure (Section 7.5.2) under the following conditions:

Method A:

(2S)-Methyl 2-N-acetylamino-5-phenylpent-4-enoate 77 (59.3 mg, 0.24mmol), DCM (10 mL), Grubbs' catalyst (19.8 mg, 24.1 μmol, 10 mol %), 50°C., 13 h, 0% conversion into dimer 60. The starting enamide 77 wasrecovered. ¹H n.m.r. spectroscopic data for olefin 77 were in agreementwith those previously reported (Section 7.11.9).

Method B:

(2S)-Methyl 2-N-acetylamino-5-phenylpent-4-enoate 77 (59.3 mg, 0.24mmol), DCM (7 mL), 2^(nd) generation Grubbs' catalyst (10.2 mg, 12.0μmol, 5 mol %), 50° C., 20 h, 44% conversion into dimer 60. ¹H n.m.r,spectroscopic data for dimer 60 were in agreement with those previouslyreported (Section 7.12.1). The stilbene byproduct 145 was observed inthe ¹H n.m.r. spectrum. ¹H n.m.r. (300 MHz, CDCl₃): 7.15 (s, 2H, CH═),7.40 (m, 4H, Arom CH), 7.55 (m, 4H, Arom CH), ortho-Arom CH peaks maskedby starting olefin 77. ¹H n.m.r. spectroscopic data for stilbene 145were in agreement with those reported in the literature.²⁶⁵

7.8.11 Dimerisation of (2S)-Methyl 2-N-Acetylaminopent-4-enoate 21a inthe presence of (2S)-Methyl 2-N-Acetylamino-5-methylhex-4-enoate 19

The mixture of olefins 21a and 19 was subjected to the conventionalcross metathesis procedure (Section 7.5.2) under the followingconditions: (2S)-Methyl 2-N-acetylaminopent-4-enoate 21a (12.7 mg, 74.2μmol), (2S)-methyl 2-N-acetylamino-5-methylhex-4-enoate 19 (14.5 mg,72.9 μmol), DCM (4 mL), Grubbs' catalyst (11.5 mg, 14.0 μmol, 20 mol %),50° C., 18 h, 100% conversion of 21a into dimer 60. ¹H n.m.r.spectroscopic data for dimer 60 were in agreement with those previouslyreported (Section 7.12.1). The prenylglycine derivative 19 was recoveredunchanged.

7.8.12 Ethenolysis of (2S)-Methyl 2-N-Acetylamino-5-methylhex-4-enoate19

The prenylglycine derivative 19 was subjected to the conventional crossmetathesis procedure (Section 7.5.4) with ethylene under the followingconditions:

Method A:

(2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate 19 (11.0 mg, 55.2μmol), ethylene (atmospheric pressure), Grubbs' catalyst (11.0 mg, 13.4μmol, 20 mol %), DCM (4 mL), 22° C., 17h, 0% conversion into 21a. ¹Hn.m.r. spectroscopy indicated the starting hex-4-enoate 19 wasrecovered.

Method B:

(2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate 19 (10.8 mg, 54.2 μmol,ethylene (60 psi), 2^(nd) generation Grubbs' catalyst (9.3 mg, 11 μmol,20 mol %), DCM (4 mL), 22° C., 19 h, 9% conversion into 21a.

Method C:

(2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate 19 (24.3 mg, 0.12mmol), ethylene (60 psi), 2^(nd) generation Grubbs' catalyst (31.1 mg,36.6 μmol, 30 mol %), DCM (5 mL), 50° C., 38 h, 32% conversion into 21a.Spectroscopic data for 21a and the recovered prenylglycine derivative 19were in agreement with those previously reported (Section 7.11.2 andSection 7.9.5 respectively).

7.8.13 Butenolysis of (2S)-Methyl 2-N-Acetylamino-5-methylhex-4-enoate19

The prenylglycine derivative 19 was subjected to the conventional crossmetathesis procedure (Section 7.5.4) with cis-2-butene under thefollowing conditions: (2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate19 (16.2 mg, 81.4 μmol), DCM (5 mL), 2^(nd) generation Grubbs' catalyst(3.5 mg, 4.1 μmol, 5 mol %), cis-2-butene (5 psi), 50° C., 14 h, 100%conversion into 81. Purification by flash chromatography (SiO₂, lightpetroleum:DCM EtOAc:MeOH, 1:2:1:0.2) gave (2S)-methyl2-N-acetylaminohex-4-enoate 81 as a brown oil (12.6 mg, 84%). GC: t_(R)(E/Z)=4.2 min, 4.4 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C.min⁻¹ to 280° C. for 6 min). ν_(max)(neat): 3284s, 2966w, 2954m, 2856w,1747s, 1658s, 1547s, 1437s, 1375s, 1217m, 1142m, 1072w, 1016w, 968m,848m cm⁻¹. ¹H n.m.r. (300 MHz, CDCl₃): δ 1.60 (dd, J=6.3, 1.2 Hz, 3H,H6), 1.95 (s, 3H, CH₃CO), 2.36-2.44 (m, 2H, H3), 3.67 (s, 3H, OCH₃),4.55 (dt, J=7.8 Hz, 5.9 Hz, 1H, H2), 5.24 (m, 1H, H5), 5.49 (m, 1H, H4),6.17 (bd, J=6.4 Hz, 1H, NH). ¹³C n.m.r. (100 MHz, CDCl₃): δ 18.1 (C6),23.3 (CH₃CO), 35.4 (C3), 52.1, 52.4 (C2, OCH₃), 124.6, 130.2 (C4, 5),169.7, 172.6 (C1, CONH). Mass Spectrum (ESI⁺, MeOH): m/z 208.1 (M+Na)⁺,C₉H₁₅NNaO₃ requires 208.1. Spectroscopic data were in agreement withthose reported in the literature.^(117,119)

An analogous cross metathesis reaction was performed using a mixture ofcis+trans-2-butene under the following conditions: (2S)-Methyl2-N-acetylamino-5-methylhex-4-enoate 19 (12.8 mg, 64.3 μmol), DCM (5mL), 2^(nd) generation Grubbs' catalyst (2.8 mg, 3.3 μmol, 5 mol %),trans+cis-2-butene (10 psi), 50° C., 16 h, <10% conversion intocrotylglycine 81.

7.8.14 Dimerisation of (2S)-Methyl 2-N-Acetylaminohex-4-enoate 81

The crotylglycine derivative 81 was subjected to the conventional crossmetathesis procedure (Section 7.5.2) under the following conditions:(2S)-Methyl 2-N-acetylaminohex-4-enoate 81 (17.0 mg, 91.9 μmol), DCM (4mL), 2^(nd) generation Grubbs' catalyst (4.2 mg, 5.0 μmol, 5 mol %), 50°C., 17 h, 100% conversion into dimer 60. The solvent was evaporatedunder reduced pressure to give the homodimer 60 as a brown oil (21.5 mg,100% crude yield). Spectroscopic data for dimer 60 were in agreementwith those previously reported (Section 7.12.1).

7.8.15 Activation of (2S)-Methyl 2-N-Benzoylamino-5-methylhex-4-enoate87

The prenylglycine derivative 87 was subjected to the conventional crossmetathesis procedure (Section 7.5.5) with cis-1,4-diacetoxy-2-butene 141under the following conditions: (2S)-Methyl2-N-benzoylamino-5-methylhex-4-enoate 87 (170 mg, 0.65 mmol), DCM (10mL), 2^(nd) generation Grubbs' catalyst (16.5 mg, 0.03 mmol, 5 mol %),cis-1,4-diacetoxy-2-butene (5 psi), 50° C., 20 h, 100% conversion into142. Purification by flash chromatography (SiO₂, light petroleum:EtOAc,1:1) gave (2S)-6-Acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 142as a dark brown oil (113 mg, 57%). ν_(max) (neat): 3333.3s; 3056.4w;3015.4w; 2943.6s; 1738.5s; 1661.5m; 1641.0s; 1605.1m; 1574.4m; 1533.3s;1487.2m; 1435.9m; 1364.1, m; 1235.9, s; 1153.8, w; 1071.6, w; 1025.6, m;969.2, m; 800.8, w; 717.9, m; 692.3,w cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ2.00, s, 3H, CH₃; 2.67, m, 2H, H3; 3.77, s, 3H, OCH₃; 4.49, d, J 4.7 Hz,2H, H6; 4.89, q, J 5.8 Hz, 1H, H2; 5.68, t, J 5.2 Hz, 2H, H4, 5; 6.75,d, J 7.4 Hz, 2H, H4, 5; 7.42, t, J 7.2 Hz, 2H, H4′, 6′; 7.50, t, J 6.4Hz, 1H, H5′; 7.78, d, J 7.1 Hz, 2H, H3′, 7′. ¹³C NMR (125 MHz, CDCl₃): δ20.92, CH₃; 35.22, C3; 52.09, OCH₃; 52.65, C2; 64.52, C6; 127.17, C3′,7′; 128.70, C4′, 6′; 128.93, C5; 129.14, C4; 131.93, C5′; 133.93, C2′;167.07, C1′; 170.80, C1″; 172.27, C1. Mass Spectrum (ESP, CH₃CN): m/z328.1 (M+Na⁺) C₁₆H₁₉NO₅Na.

7.8.16 Synthesis of (2S,7S)-dimethyl2-N-acetylamino-7-N-benzoylamino-octa-4-enedioate 143

2-Acetylamino-7-benzoylamino-oct-4-enedioic acid dimethyl ester 143 wassynthesised using standard solution-phase metathesis conditions (referto Section 7.5.2): 6-Acetoxy-2-benzoylamino-4-hexenoic acid methyl ester142 (50 mg, 0.16 mmol), dichloromethane (10 mL), second generationGrubbs' catalyst (5 mol %, 7 mg, 8 μmol),methyl-2-acetylamino-4-pentenoate 21a (168 mg, 0.98 mmol), 50° C., 18 h.The desired compound was obtained as a brown oil and purified via columnchromatography (SiO₂; EtOAc:hexane; 2:1). ¹H NMR (500 MHz, CDCl₃,mixture of isomers (1:1.2)): δ 1.95, s (major isomer) and 1.97, s (minorisomer), 3H, CH₃; 2.42-2.70, m, 4H, H3, 6; 3.62, s (minor isomer), 3.64,s (major isomer), 3.78, s (minor isomer) and 3.79, s (major isomer), 6H,2×OCH₃; 4.63-4.66, m, 1H, H2; 4.85-4.91, m, 1H, H7; 5.35-5.49, m, 2H,114, 5; 6.20, d, J 7.7 Hz (major isomer) and 6.34, d, J 7.5 Hz, 1H, NH(minor isomer); 6.87, t, J 7.55 Hz, 1H, NH; 7.44, t, J 7.1 Hz, 2H, H4′,6′; 7.50, t, J 6.9 Hz, 1H, H5′; 7.84, t, J 7.9 Hz, 2H, H3′, 7′. ¹³C NMR(75 MHz, CDCl₃, mixture of isomers (1:1.2)): δ 22.83, CH₃; 34.84, 35.05,35.38 and 35.73, C3, 6; 51.51 and 51.55, C2; 52.35, 52.46, 52.53, 52.60and 52.66, C7, 2×OCH₃; 127.18 and 127.22, C3′, 7′; 128.57 and 128.62,C4′, 6′; 128.88 and 129.00, C4, 5; 131.86 and 131.91, C5′; 133.71, C2′;167.06, COPh; 170.03 and 170.11, COMe; 172.20, 172.21, 172.24 and172.43, 2×COOMe. Mass Spectrum (ESI⁺, CH₃OH): m/z 399.2 (M+Na⁺)C₁₉H₂₄N₂O₆Na.

7.9 Wilkinson's Hydrogenation of Olefinic Substrates 7.9.1(2S,7S)-Dimethyl 2,7-N,N′-Diacetylaminooctanedioate 71

(2S,7S)-Dimethyl 2,7-N,N′-diacetylaminooct-4-enedioate 60 was subjectedto the general Wilkinson's hydrogenation procedure (Section 7.4.4) underthe following conditions: Dimer 60 (25.0 mg, 79.6 μmol), benzene (5 mL),Wilkinson's catalyst, 60 psi, 22° C., 4 h. At the end of the reactionperiod, the solvent was evaporated under reduced pressure and theresulting oil was purified by flash chromatography (SiO₂, EtOAc) toafford the saturated product 71 as a brown oil (25.0 mg, 99%). GC:t_(R)=14.4 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min⁻¹ to280° C. for 6 min). ν_(max) (neat): 3426bm, 3055w, 2932m, 2857w, 2360w,1741s, 1666s, 1543w, 1438m, 1375w, 1266s, 1177w, 1120w, 896w, 738w, 702wcm⁻¹. ¹H n.m.r. (400 MHz, CDCl₃): δ 1.30-1.40 (m, 4H, H4, 5), 1.82-1.90(m, 4H, H3, 6), 2.02 (s, 6H, CH₃CO), 3.74 (s, 6H, OCH₃), 4.56-4.63 (m,2H, H2, 7), 6.16 (bd, J=7.5 Hz, 2H, NH). ¹³C n.m.r. (100 MHz, CDCl₃): δ23.3 (CH₃CO), 24.7 (C4, 5), 32.3 (C3, 6), 52.0 (C2, 7), 52.5 (OCH₃),170.0, 173.1 (C1, 8, CONH). HRMS MeOH). Found: m/z 339.1531 (M+Na)⁺,C₁₄H₂₄N₂NaO₆ requires 339.1532.

7.9.2 (2S,7S)-Dimethyl 2,7-N,N′-Dibenzoylaminooctanedioate 72

(2S,7S)-Dimethyl 2,7-N,N′-dibenzoylaminoocta-4-enedioate 69 wassubjected to the general Wilkinson's hydrogenation procedure (Section7.4.4) under the following conditions: Dimer 69 (20.0 mg, 45.7 μmol,benzene (5 mL), Wilkinson's catalyst, 60 psi, 22° C., 4 h. At the end ofthe reaction period, the solvent was evaporated under reduced pressureand the resulting oil was purified by flash chromatography (SiO₂, EtOAc)to afford the saturated product 72 as a brown oil (20.0 mg, 100%). GC:t_(R) 17.2 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min⁻¹ to280° C. for 6 min). ν_(max) (neat): 3055m, 2986w, 2955w, 1741s, 1662s,1603w, 1580w, 1518m, 1486m, 1438s, 1359w, 1286s, 1182m, 1120m, 1028w,896m cm⁻¹. ¹H n.m.r. (400 MHz, CDCl₃): δ 1.35-1.53 (m, 4H, H4, 5),1.80-2.02 (m, 4H, H3, 6), 3.78 (s, 6H, OCH₃), 4.82 (dt, J=7.3, 5.4 Hz,2H, H2, 7), 6.73 (bd, J=7.4 Hz, 2H, NH), 7.40-7.49 (m, 6H, H3′, 4′, 5′),7.78-7.82 (m, 4H, H2′, 6′). ¹³C n.m.r. (100 MHz, CDCl₃): δ 24.9 (C4, 5),32.6 (C3, 6), 52.5, 52.7 (C2, OCH₃), 127.2 (C2′, 6′), 128.6 (C3′, 5′),131.9 (C4′), 134.1 (C1′), 167.2, 173.2 (C1, 8, CONH). HRMS (ESI⁺, MeOH).Found: m/z 463.1842 (M+Na)⁺, C₂₄H₂₈N₂NaO₆ requires 463.1845.

7.9.3 Wilkinson's Hydrogenation of (2Z)-Methyl2-N-Acetylamino-5-phenylpenta-2,4-dienoate 76

The dienamide 76 was subjected to the general Wilkinson's hydrogenationprocedure (Section 7.4.4) under the following conditions: (2Z)-Methyl2-N-acetylamino-5-phenylpenta-2,4-dienoate 76 (11.5 mg, 46.9 μmol),benzene (5 mL), Wilkinson's catalyst, 50 psi H₂, 22° C., 4 h, 99% yield(mass recovery) of a 1:4 mixture of 77:79 as a brown oil. GC: t_(R)=10.8min 79, 13.9 min 77 (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C.min⁻¹ to 280° C. for 6 min). ¹H n.m.r. spectroscopic datafor olefin 77were in agreement with those previously reported (Section 7.11.9).Hydrogenation of the mixture using identical conditions led to 100%conversion into 79 (41.2 mg, 100% crude yield). ν_(max) (neat): 3262w,3054m, 2956m, 1736s, 1676s, 1509m, 1438s, 1372w, 1265s, 1174w, 1120m,1028w, 738s, 700w cm⁻¹. ¹H n.m.r. (300 MHz, CDCl₃): δ 1.53-1.65 (m, 4H,H3, 4), 1.94 (s, 3H, CH₃CO), 2.52-2.59 (rn, 2H, H5), 3.65 (s, 3H, OCH₃),4.59 (m, 1H, H2), 5.90 (bd, J=7.2 Hz, 1H, NH), 7.07-7.29 (m, 5H, AromCH). ¹³C n.m.r. (75 MHz, CDCl₃): δ 23.3 (CH₃CO), 27.2 (C4), 32.3 (C3),35.5 (C5), 52.1, 52.5 (C2, OCH₃), 126.1, 128.5, 132.2 (Arom CH), 141.7,(Arom C), 169.9, 173.2 (C1, CONH). Mass Spectrum (ESI⁺, MeOH): m/z 272.2(M+Na)⁺, C₁₄H₁₉NNaO₃ requires 272.1.

7.9.4 Wilkinson's Hydrogenation of (2S)-Methyl2-N-Acetylamino-5-methylhex-4-enoate 19

(2S)-Methyl 2-N-acetylamino-5-methylhex-4-enoate 19 was subjected to thegeneral Wilkinson's hydrogenation procedure (Section 7.4.4) under thefollowing conditions: Hex-4-enoate derivative 19 (11.3 mg, 56.8 μmol),benzene (4 mL), Wilkinson's catalyst, 50 psi, 22° C., 4 h. At the end ofthe reaction period, the solvent was evaporated under reduced pressureto afford a brown oil (12.5 mg). ¹H n.m.r. spectroscopy indicated thereaction gave only 6% conversion into the saturated product 80; 94% ofthe starting prenylglycine derivative 19 was recovered. ¹H n.m.r. (300MHz, CDCl₃): Hexanoate 80: δ 0.87 (d, J=6.6 Hz, 6H, H6), 1.09-1.28 (m,2H, H4), 1.54 (h, J=6.7 Hz, 1H, H5), 1.61-1.71 (m, 2H, H3), 2.03 (s, 3H,CH₃CO), 3.75 (s, 3H, OCH₃), 4.60 (dt, J=7.8 Hz, 5.5 Hz, 1H, H2), 5.96(bd, J=7.8 Hz, 1H, NH).

Experimental for Chapter 5 7.10 Synthesis of Olefinic Substrates 7.10.1(2S)-Methyl 2-N-(p-Nitrobenzoyl)aminopent-4-enoate 83

A solution of p-nitrobenzoyl chloride 89 (1.21 g, 6.54 mmol) in amixture of DCM:Et₂O (2:1, 15 mL) was added dropwise to a stirredsolution of methyl 2-aminopent-4-enoate hydrochloride 51 (0.98 g, 5.94mmol) and Et₃N (1.80 mL, 13.0 mmol) in a mixture of DCM:Et₂O (2:1, 15mL) at 0° C. The reaction mixture was allowed to warm to roomtemperature and stirred for 20 h. The mixture was acidified with diluteHCl solution (1 M, pH 2) and extracted with DCM (3×20 mL). The combinedorganic extract was washed with distilled water (20 mL), dried (MgSO₄)and evaporated under reduced pressure to afford the titled compound 83as an off-white solid (1.63 g, 99%), m.p. 99-100° C. Spectroscopic dataindicated the crude product 83 did not require purification and was useddirectly in the subsequent reaction (Section 7.15.1). GC: t_(R)=12.20min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min⁻¹ to 280° C.for 6 min). [α]_(D) ²²+29.9° (c=0.37, CHCl₃). ν_(max) (neat): 3293w,2954m, 2839m, 1725s, 1641m, 1602w, 1538w, 1529w, 1456s, 1377s, 1256m,1160m, 1118w, 1066w, 998m, 972m, 941w, 841m cm⁻¹. ¹H n.m.r. (300 MHz,CDCl₃): δ 2.65-2.76 (m, 2H, H3), 3.82 (s, 3H, OCH₃), 4.90 (dt, J=5.6,7.5 Hz, 1H, H2), 5.14-5.30 (m, 2H, H5), 5.75 (m, 1H, H4), 6.73 (bd,J=6.6 Hz, 1H, NH), 7.95 (d, J=7.7 Hz, 2H, H2′, 6′), 8.30 (d, J=7.6 Hz,2H, H3′, 5′). ¹³C n.m.r. (75 MHz, CDCl₃): δ 36.6 (C3), 52.4, 52.9 (C2,OCH₃), 119.9 (C5), 124.0 (C3′, 5′), 128.4 (C2′, 6′), 132.0 (C4), 139.6(C1′), 150.0 (C4′), 165.1 (C1), 172.1 (CONH). HRMS (ESI⁺, MeOH). Found:m/z 279.0977 (M+H)⁺, C₁₃H₁₅N₂O₅ requires 279.0981; m/z 301.0798 (M+Na)⁺,C₁₃H₁₄N₂NaO₅ requires 301.0800.

7.10.2 (2S)-Methyl 2-N-Acetylamino-5-methylhex-4-enoate 19

The preparation of (2S)-methyl 2-N-acetylamino-5-methylhex-4-enoate 19via asymmetric hydrogenation of the dienoate 20 has been previouslydescribed (Section 7.9.5).

7.10.3 (2Z)-Methyl 2-N-Benzoylamino-5-methylhexa-2,4-dienoate 82

The dienamide 82 was prepared according to a procedure described by Teohet al.^(118,119) Tetramethylguanidine (3.40 mL, 27.1 mmol) andhydroquinone (3.0 mg) were added to a solution of methyl2-N-benzoylamino-2-(dimethoxyphosphinyl)-acetate 64 (6.10 g, 20.3 mmol)in THF (120 mL) at −78° C. After 30 min, 3-methyl-2-butenal 40 (2.40 mL,24.9 mmol) was added and the mixture was stirred at −78° C. for 2 h,warmed to 25° C. and allowed to react an additional 16 h. The mixturewas diluted with DCM (150 mL) and washed with dilute HCl solution (1 M,2×70 mL), CuSO₄ solution (1 M, 2×70 mL), saturated NaHCO₃ solution (2×70mL) and saturated NaCl solution (1×70 mL). The organic extract was dried(MgSO₄) and evaporated under reduced pressure to give the crude product82 as a yellow oil (5.37 g). Purification by flash chromatography (SiO₂,light petroleum:EtOAc, 2:1) furnished the pure dienamide 82 as anoff-white solid (3.84 g, 73%), m.p. 98-99° C. GC: t_(R)=11.00 min (GCcolumn 30QC5/BPX5, 150° C. for 1 min, 10° C. min⁻¹ to 280° C. for 6min). ν_(max) (KBr): 3286m, 2991w, 2948w, 1716s, 1649s, 1601w, 1579w,1524s, 1489s, 1436m, 1389w, 1331m, 1286m, 1254s, 1207m, 1190w, 1162w,1135w, 1087s, 1048w, 996w, 977w, 931w, 864m, 802m, 760m, 739m, 710s,688w, 677w, 630w, 614w, 585w cm⁻¹. ¹H n.m.r. (400 MHz, CDCl₃): δ 1.87(s, 3H, H6), 1.91 (d, J=0.7 Hz, 3H, CH₃C—), 3.78 (s, 3H, OCH₃), 6.03 (dwith fine splitting, J=11.9 Hz, 1H, H4), 7.41 (d, J=11.8 Hz, 1H, H3),7.43-7.47 (m, 2H, H3′, 5′), 7.53 (m, 1H, H4′), 7.63 (bs, 1H, NH),7.89-7.90 (m, 2H, H2′, 6′). ¹³C n.m.r. (100 MHz, CDCl₃): δ 19.3 (CH₃C═),27.1 (C6), 52.5 (OCH₃), 121.0 (C4), 121.2 (C5), 127.6 (C2′, 6′), 128.9(C3′, 55, 129.9 (C3), 132.0 (C4′), 134.1 (C2), 147.2 (C1′), 166.0, 166.1(C1, CONH). HRMS (ESI⁺, MeOH). Found: m/z 260.1282 (M+H)⁺, C₁₅H₁₈NO₃requires 260.1287; m/z 282.1099 (M+Na)⁺, C₁₅H₁₇NNaO₃ requires 282.1106.

7.11 Metathesis Reactions with Olefinic Substrates 7.11.1(2S,7S)-Dimethyl 2,7-N,N′-Di[(p-nitrobenzoyl)amino]oct-4-enedioate 90

Method A:

The dimer 90 was prepared via the conventional cross metathesisprocedure (Section 7.5.2) under the following conditions: (2S)-Methyl2-N-(p-nitrobenzoyl)aminopent-4-enoate 83 (43.5 mg, 0.16 mmol), DCM (3mL), Grubbs' catalyst (26.0 mg, 31.6 μmol, 20 mol %), 50° C., 14 h, 100%conversion into dimer 90. The reaction mixture was evaporated underreduced pressure to give the homodimer 90 as a brown oil (69.7 mg, 100%crude yield).

Method B:

The dimer 90 was also prepared and purified from an analogous reactionusing 2^(nd) generation Grubbs' catalyst under the following conditions:(2S)-Methyl 2-N-(p-nitrobenzoyl)aminopent-4-enoate 83 (86.3 mg, 0.31mmol), DCM (4 mL), 2^(nd) generation Grubbs' catalyst (13.6 mg, 16.0μmol, 5 mol %), 50° C., 12 h, 100% conversion into dimer 90.Purification by flash chromatography (SiO₂, light petroleum:EtOAc:DCM,4:2:1) gave the pure dimer 90 as a pale brown solid (74.0 mg, 90%), m.p.90-92° C. GC: t_(R) (E/Z)=16.1 min, 16.2 min (GC column 30QC5/BPX5, 150°C. for 1 min, 10° C. min⁻¹ to 280° C. for 6 min). [α]_(D) ²²+20.0°(c=0.21, CHCl₃). ν_(max) (neat): 3365m, 3057w, 2957w, 2854w, 1728s,1667s, 1602m, 1525s, 1487m, 1437m, 1348s, 1267m, 1227m, 1174w, 1157w,1110w, 1014m, 974m, 869m, 874m, 737s, 718s em′. ¹H n.m.r. (400 MHz,CDCl₃): δ 2.60-2.64 (m, 4H, H3, 6), 3.70 (s, 6H, OCH₃), 4.88 (apparentq, J=5.9 Hz, 2H, H2, 7), 5.49-5.53 (m, 2H, H4, 5), 7.11 (bd, J=7.4 Hz,2H, NH), 8.02 (d, J=8.7 Hz, 4H, H2′, 6′), 8.21-8.29 (m, 4H, H3′, 5′).¹³C n.m.r. (100 MHz, CDCl₃): δ 35.0 (C3, 6), 52.8, 52.8 (C2, 7, OCH₃),123.8 (C3′, 5′), 128.8, 128.9 (C2′, 6′, C4, 5), 139.2 (C1′), 149.9(C4′), 165.2 (C1, 8), 172.1 (CONH). HRMS (ESI⁺, MeOH). Found: m/z529.1560 (M+H)⁺, C₂₄H₂₅N₄O₁₀ requires 529.1571; m/z 551.1379 (M+Na)⁺,C₂₄H₂₄N₄NaO₁₀ requires 551.1390.

7.11.2 Attempted Dimerisation of (2Z)-Methyl2-N-Benzoylamino-5-methylhexa-2,4-dienoate 82

The dienamide 82 was subjected to the conventional cross metathesisprocedure (Section 7.5.2) under the following conditions: (2Z)-Methyl2-N-benzoylamino-5-methylhexa-2,4-dienoate 82 (30.7 mg, 0.12 mmol), DCM(5 mL), 2^(nd) generation Grubbs' catalyst (5.1 mg, 6.0 μmol, 5 mol %),50° C., 15 h, 0% conversion into dimer 84. The dienamide 82 wasrecovered unchanged. ¹H n.m.r. spectroscopic data for the recovereddienamide 82 were in agreement with those previously reported (Section7.14.3).

7.11.3 Attempted Butenolysis of (2Z)-Methyl2-N-Benzoylamino-5-methylhexa-2,4-dienoate 82

The dienamide 82 was subjected to the conventional cross metathesisprocedure (Section 7.5.4) with cis-2-butene under the followingconditions: (2Z)-Methyl 2-N-benzoylamino-5-methylhexa-2,4-dienoate 82(39.3 mg, 0.15 mmol), DCM (5 mL), cis-2-butene (15 psi), 2^(nd)generation Grubbs' catalyst (6.6 mg, 7.8 μmol, 5 mol %), 50° C., 14 h,0% conversion into 86. The dienamide 82 was recovered unchanged. ¹Hn.m.r. spectroscopic data for the recovered dienamide 82 were inagreement with those previously reported (Section 7.14.3).

7.12 Activation of Dormant Olefins 7.12.1 Butenolysis of (2Z)-Methyl2-N-Acetylamino-5-methylhex-4-enoate 19

The activation of prenylglycine 19 via butenolysis (Section 7.5.4) togive the crotylglycine derivative 81 has been previously described(Section 7.12.13).

7.12.2 (2S)-Methyl 2-N-Benzoylamino-5-methylhex-4-enoate 87

The dienamide 82 was subjected to the general asymmetric hydrogenationprocedure (Section 7.4.3) under the following conditions: (2Z)-Methyl2-N-benzoylamino-5-methylhexa-2,4-dienoate 82 (26.1 mg, 0.10 mmol), MeOH(5 mL), Rh(I)—(S,S)-Et-DuPHOS, 75 psi, 22° C., 3 h. At the end of thereaction period, the solvent was evaporated under reduced pressure andthe residue was purified by flash chromatography (SiO₂, EtOAc) to givethe prenylglycine derivative 87 as a pale yellow oil (23.9 mg, 91%).HPLC: t_(R)=6.20 min (Chiralcel OJ column, 1.0 mL min⁻¹, detection at254 nm, 5% EtOH:95% hexane). [α]_(D) ²²+53.0° (c=1.19, CHCl₃). ν_(max)(neat): 3334m, 2953w, 1744s, 1645s, 1603w, 1580w, 1538s, 1489m, 1437m,1353w, 1274w, 1211w, 1175w, 1095w, 1031w, 736w, 714w, 693w cm⁻¹. ¹Hn.m.r. (300 MHz, CDCl₃): δ 1.61 (d, J=0.5 Hz, 3H, CH₃C═), 1.71 (d, J=1.0Hz, 3H, H6), 2.52-2.76 (m, 2H, H3), 3.77 (s, 3H, OCH₃), 4.85 (dt, J=7.7,5.5 Hz, 1H. H2), 5.08 (m, 1H, H4), 6.65 (bd, J=6.9 Hz, 1H NH), 7.41-7.47(m, 2H, H3′, 5), 7.51 (m, 1H, H4′), 7.76-7.79 (m, 2H, H2′, 6′). ¹³Cn.m.r. (75 MHz, CDCl₃): δ 18.0 (CH₃C═), 26.0 (C6), 30.9 (C3), 52.5, 52.6(C2, OCH₃), 117.6 (C4), 127.2 (C2′, 6′), 128.7 (C3′, 5′), 131.8 (C4′),134.3 (C5), 136.8 (C1′), 167.0, 172.8 (C1, CONH). HRMS (ESI⁺, MeOH).Found: m/z 262.1441 (M+H)⁺, C₁₅H₂₀NO₃ requires 262.1443; m/z 284.1256(M+Na)⁺, C₁₅H₁₉NNaO₃ requires 284.1263.

(2R)-Methyl 2-N-benzoylamino-5-methylhex-4-enoate 87

The dienamide 82 was subjected, to the general asymmetric hydrogenationprocedure (Section 7.4.3) under the following conditions: (2Z)-Methyl2-N-benzoylamino-5-methylhexa-2,4-dienoate 82 (80.1 mg, 0.31 mmol), MeOH(7 mL), Rh(I)—(R,R)-Et-DuPHOS, 75 psi, 22° C., 3 h. At the end of thereaction period, the solvent was evaporated under reduced pressure andthe residue was purified by flash chromatography (SiO₂, EtOAc) to givethe prenylglycine derivative 87 as a yellow oil (78.2 mg, 97%). HPLC:t_(R)=5.90 min (Chiralcel OJ column, 1.0 mL min^(−I), detection at 254nm, 5% EtOH:95% hexane). [α]_(D) ²²−53.4° (c=0.98, CHCl₃). Spectroscopicdata were in agreement with those previously reported for the(S)-enantiomer.

7.12.3 (2S)-Methyl 2-N-Benzoylaminohex-4-enoate 88

The enamide 87 was subjected to the conventional cross metathesisprocedure (Section 7.5.5) with cis-2-butene under the followingconditions: (2S)-Methyl 2-N-benzoylamino-5-methylhex-4-enoate 87 (90.0mg, 0.35 mmol), DCM (5 mL), cis-2-butene (15 psi), 2^(nd) generationGrubbs' catalyst (14.6 mg, 17.2 μmol, 5 mol %), 50° C., 12 h, 100%conversion into 88. The reaction mixture was evaporated under reducedpressure to give the crotylglycine derivative 88 as a brown oil (101 mg,100% crude yield). GC: t_(R) (E/Z)=9.68 min, 9.93 min (GC Column30QC5/BPX5, 150° C. for 1 min, 10° C. min⁻¹ to 280° C. for 6 min).ν_(max) (neat): 3337bm, 3057w, 2954m, 2856w, 1743s, 1652s, 1603w, 1580w,1532s, 1488m, 1438m, 1360w, 1266s, 1217w, 1180w, 1116w, 1031m, 969w,896w, 801w, 738s, 638w cm⁻¹. ¹H n.m.r. (300 MHz, CDCl₃): δ 1.66 (dd,J=6.4, 1.4 Hz, 3H, H6), 2.52-2.66 (m, 2H, H3), 3.77 (s, 3H, OCH₃), 4.82(apparent dd, J=7.6, 5.7 Hz, 1H, H2), 5.33 (m, 1H, H5), 5.63 (m, 1H,H4), 6.66 (bd, J=7.0 Hz, 1H, NH), 7.43 (t, J=7.0 Hz, 2H, H3′, 5′), 7.50(m, 1H, H4′), 7.78 (d, J=7.1 Hz, 2H, H2′, 6′). ¹³C n.m.r. (100 MHz,CDCl₃): δ 18.0 (C6), 35.4 (C3), 52.4, 52.5 (C2, OCH₃), 124.5 (C5), 127.1(CT, 6′), 128.6 (C3′, 5′), 130.2 (C4), 131.7 (C4′), 134.1 (C1′), 166.9,172.5 (C1, CONH). HRMS (ESI⁺, MeOH). Found: m/z 248.1284 (M+H)⁺,C₁₄H₁₈NO₃ requires 248.1287; m/z 270.1098 (M+Na)⁺, C₁₄H₁₇NNaO₃ requires270.1106.

7.12.4 Dimerisation of (2S)-Methyl 2-N-Acetylaminohex-4-enoate 81

The dimerisation of crotylglycine 81 using the conventional crossmetathesis procedure has been previously described (Section 7.12.14).

7.12.5 Dimerisation of (2S)-Methyl 2-N-Benzoylaminohex-4-enoate 88

The enamide 88 was subjected to the conventional cross metathesisprocedure under the following conditions: (2S)-Methyl2-N-benzoylaminohex-4-enoate 88 (89.6 mg, 0.36 mmol), DCM (5 mL), 2^(nd)generation Grubbs' catalyst (15.3 mg, 18.0 μmol, 5 mol %), 50° C., 17 h,100% conversion into dimer 69. The reaction mixture was evaporated underreduced pressure to afford the homodimer 69 as a brown oil (106 mg, 100%crude yield). Spectroscopic data for dimer 69 were in agreement withthose previously reported (Section 7.12.2).

7.13 Wilkinson's Hydrogenation Reactions 7.13.1 Wilkinson'sHydrogenation of (2Z)-Methyl 2-N-Benzoylamino-5-methylhexa-2,4-dienoate82

Method A:

The dienamide 82 was subjected to the general Wilkinson's hydrogenationprocedure (Section 7.4.4) under the following conditions: (2Z)-Methyl2-N-benzoylamino-5-methylhexa-2,4-dienoate 82 (47.0 mg, 0.18 mmol),benzene (5 mL), Wilkinson's catalyst, 50 psi, 22° C., 4 h. The dienamide82 was recovered unchanged. ¹H n.m.r. spectroscopic data for therecovered dienamide 83 were in agreement with those previously reported(Section 7.14.3).

7.13.2 (2S,7S)-Dimethyl 2,7-N,N′-Di(p-nitrobenzoyl)aminooctanedioate 91

(2S,7S)-Dimethyl 2,7-N,N′-di(p-nitrobenzoyl)aminoocta-4-enedioate 90 wassubjected to the general Wilkinson's hydrogenation procedure (Section7.4.4) under the following conditions: Dimer 90 (20.6 mg, 0.04 mmol),THF:^(t)BuOH (1:1, 5 mL), Wilkinson's catalyst, 15 psi H₂, 22° C., 14 h.At the end of the reaction period, the solvent was evaporated underreduced pressure to afford the product 91 as a brown oil. Purificationby flash chromatography (SiO₂, light petroleum:EtOAc:DCM, 1:1:1) gavethe pure dimer 91 as an off-white solid (13.8 mg, 67%), m.p. 117-119° C.GC: t_(R)=16.8 min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C.min⁻¹ to 280° C. for 6 min). ν_(max) (neat): 3304w, 2932w, 1740s, 1637s,1603m, 1528s, 1438w, 1348m, 1265s, 1109w cm⁻¹. ¹H n.m.r. (400 MHz,CDCl₃): δ 1.39-1.54 (m, 4H, H4, 5), 1.74-2.04 (m, 4H, H3, 6), 3.81 (s,6H, OCH₃), 4.82 (dt, J=7.3, 5.4 Hz, 2H, H2, 7), 6.85 (bd, J=7.4 Hz, 2H,NH), 7.96 (d, J=8.8 Hz, 4H, H2′, 6′), 8.28 (d, J=8.7 Hz, 4H, H3′, 5′).¹³C n.m.r. (100 MHz, CDCl₃): δ 24.7 (C4, 5), 32.4 (C3, 6), 52.7, 52.9(C2, OCH₃), 124.0 (C2′, 6′), 128.5 (C3′, 5′), 139.5 (C1′), 150.0 (C4′),165.3, 172.8 (C1, 8, CONH). HRMS (ESI⁺, MeOH). Found: m/z 553.1550(M+Na)⁺, C₂₄H₂₆N₄NaO₁₀ requires 553.1547.

Experimental for Section 6 7.14 Synthesis of Non-ProteinaceousFmoc-Amino Acids

Peptide sequences are represented by structural diagrams andthree-letter codes of constituent amino acids. Synthetic amino acidsallylglycine, crotylglycine and prenylglycine are represented by Hag,Crt and Pre respectively. Procedures for the preparation of theFmoc-protected olefinic amino acids:(2S)-2-N-Fluorenylmethoxy-carbonylaminopent-4-enoic acid (Fmoc-L-Hag-OH)96, (2S)-2-N-fluorenylmethoxy-carbonylaminohex-4-enoic acid(Fmoc-L-Crt-OH) 100 and(2S)-2-N-fluorenyl-methoxycarbonylamino-5-methylhex-4-enoic acid(Fmoc-L-Pre-OH) 92, are detailed below.

7.14.1 2-N-Fluorenylmethoxycarbonylaminopent-4-enoic acid 96(Fmoc-Hag-OH)

The allylglycine derivative 96 was prepared according to the proceduredescribed by Paquet.²³⁰ Fmoc-OSu (14.60 g, 43.3 mmol) was added tostirred solution of L-allylglycine (5.00 g, 43.5 mmol) and NaHCO₃ (18.20g, 0.22 mol) in a mixture of acetone:water (200 mL). The resultant whitesuspension was stirred at room temperature and after 20 h, t.l.c.analysis (SiO₂, light petroleum:EtOAc; 1:1) showed the absence ofstarting material. The reaction mixture was acidified with concentratedHCl (pH 2) and the acetone was removed under reduced pressure. Theresultant suspension was extracted into DCM (3×75 mL) and the combinedorganic extract was washed with dilute HCl solution (1 M, 2×50 mL),water (2×50 mL), dried (MgSO₄) and evaporated under reduced pressure toafford the titled Fmoc-amino acid 96 as a colourless solid (14.01 g,96%), m.p. 137-138° C. (lit.²⁶⁶ 134-136° C.). ν_(max) (KBr): 3484s,3198bs, 3085m, 2967m, 2923m, 1723s, 1644m, 1525s, 1478w, 1449s, 1396m,1340m, 1233s, 1189s, 1099m, 1048s, 998w, 966w, 943m, 924w, 850m, 781m,761s, 740m, 648w, 623m, 582m, 560w, 540m, 424w cm⁻¹. ¹H n.m.r. (400 MHz,CDCl₃): δ 2.52-2.70 (2.34-2.49) (m, 2H, H3), 4.23 (t, J=6.9 Hz, 1H,H9′), 4.42 (4.30) (d, J=6.9 Hz, 2H, CH₂O), 4.52 (m, 1H, H2), 5.13-5.23(m, 2H, H5), 5.31 (5.87) (bd, J=7.8 Hz, 1H, NH), 5.75 (m, 1H, H4), 6.63(bs, 1H, OH), 7.31 (td, J=7.4, 0.8 Hz, 2H, H2′, 7′), 7.38 (t, J=7.4 Hz,2H, H3′, 6′), 7.52-7.63 (m, 2H, H1′, 8′), 7.76 (d, J=7.5 Hz, 2H, H4′,5′), one exchangeable proton (OH) not observed. ¹³C n.m.r. (100 MHz,CDCl₃): δ 36.7 (C3), 47.5 (C9′), 53.4 (C2), 68.1 (CH₂O), 122.0 (C5),120.1 (C2′, 7′), 125.4 (C3′, 6′), 127.9 (C1′, 8′), 128.0 (C4′, 5′),131.1 (C4), 141.7 (C8′a, 9′a), 144.0 (C4′a, 4′b), 156.3 (OCONH), 176.4(C1). Mass Spectrum (ESI+, MeOH): m/z 338.4 (M+H)⁺, C₂₀H₂₀NO₄ requires338.1; 360.3 (M+Na)⁺, C₂₀H₁₉NNaO₄ requires 360.1. Spectroscopic datawere in agreement with those reported in the literature.²⁶⁶

7.14.2 2-N-Fluorenylmethoxycarbonylaminohex-4-enoic acid 100(Fmoc-Crt-OH)

A solution of (2S)-methyl 2-N-acetylaminohex-4-enoate 81 (1.30 g, 7.05mmol) in dilute HCl (1 M, 8 mL) was heated at reflux for 21 h. Thereaction mixture was evaporated under reduced pressure to give2-aminohex-4-enoic acid hydrochloride salt (L-crotylglycine.HCl) 101 asa pale brown solid (1.17 g, 100%), m.p. 212-214° C. ν_(max) (KBr):3500bs, 2965m, 2358s, 1731s, 1651m, 1455m, 901m cm⁻¹. ¹H n.m.r. (300MHz, CD₃OD): δ 1.69 (d, J=5.3 Hz, 3H, H6), 2.51-2.74 (m, 2H, H3), 3.99(m, 1H, H2), 5.42 (m, 1H, H5), 5.73 (m, 1H, H4), exchangeable protons(NH and OH) not observed. ¹³C n.m.r. (75 MHz, CD₃OD): δ 18.7 (C6), 35.1(C3), 48.7 (C2), 124.6 (C5), 133.6 (C4), 174.3 (C1). Mass Spectrum(ESI+, MeOH): m/z 130.1 (M+H)⁺, C₆H₁₂NO₂ requires 130.1.

2-N-Fluorenylmethoxycarbonylaminohex-4-enoic acid 100 was preparedaccording to the procedure described by Paquet.²³⁰ Fmoc-OSu (2.36 g,7.00 mmol) was added to a stirred suspension of L-crotylglycine.HCl 101(1.16 g, 7.03 mmol) and NaHCO₃ (2.95 g, 35.0 mmol) in a mixture ofacetone:water (1:1, 30 mL) The resultant suspension was stirred at roomtemperature for 15 h. The reaction mixture was then acidified withconcentrated HCl (pH 2) and the acetone was removed under reducedpressure. The resultant suspension was extracted into DCM (3×25 mL) andthe combined organic extract was washed with dilute HCl solution (1 M,2×25 mL), water (2×25 mL), dried (MgSO₄) and evaporated under reducedpressure to afford the titled Fmoc-amino acid 100 as colourless solid(1.91 g, 78%), m.p. 119-121° C. (KBr): 3390bm, 3033m, 2961s, 2357w,1730s, 1651w, 1505w, 1450w, 1395w, 850w cm⁻¹. ¹H n.m.r. (300 MHz,CDCl₃): δ 1.67 (d, J=6.2 Hz, 3H, H6), 2.37-2.69 (m, 2H, H3), 4.23 (t,J=6.8 Hz, 1H, H9′), 4.42-4.48 (m, 3H, CH₂O, H2), 5.30-5.37 (m, 2H, H5,NH), 5.61 (m, 1H, H4), 7.31 (td, J=7.2, 1.3 Hz, 2H, H2′, 7′), 7.34 (td,J=7.4, 1.5 Hz, 2H, H3′, 6′), 7.60 (d, J=7.3 Hz, 2H, H1′, 8′), 7.74 (d,J=7.0 Hz, 2H, H4′, 5′), one exchangeable proton (OH) not observed. ¹³Cn.m.r. (75 MHz, CDCl₃): δ 16.7 (C6), 34.1 (C3), 46.2 (C9′), 52.3 (C2),66.2 (CH₂O), 118.9 (C5), 123.0 (C2′, 7′), 124.6 (C3′, 6′), 125.2 (C1′,8′), 127.5 (C4′, 5′), 129.7 (C4), 140.3 (C8′a, 9′a), 142.7 (C4′a, 4′b),154.9 (OCONH), 175.0 (C1). Mass Spectrum (ESI+, MeOH): m/z 352.1 (M+H)⁺,C₂₁H₂₂NO₄ requires 352.2, Spectroscopic data were in agreement withthose reported in the literature.¹⁴⁶

7.14.3 2-N-Fluorenylmethoxycarbonylamino-5-methylhex-4-enoic acid 92(Fmoc-Pre-OH)

The allylglycine derivative 96 was subjected to the conventional crossmetathesis procedure with 2-methyl-2-butene (Section 0) under thefollowing conditions: 2-N-Fluorenylmethoxycarbonylaminopent-4-enoic acid96 (200 mg, 0.59 mmol), DCM (7 mL), 2′ generation Grubbs' catalyst (26.0mg, 30.6 μmol, 5 mol %), 2-methyl-2-butene (1 mL, 10 psi), 50° C., 12 h,100% conversion into 92. The reaction mixture was evaporated underreduced pressure to give the prenylglycine derivative 92 as a brown oil(245 mg, 100% crude yield). ν_(max) (neat): 3426w, 3324w, 3066w, 2932m,1716s, 1514m, 1478w, 1450m, 1378w, 1338m, 1265m, 1220w, 1106w, 1057m,910m, 855w, 759w, 738s, 704w, 648w, 621 w cm⁻¹. ¹H n.m.r. (400 MHz,CDCl₃): δ 1.63 (s, 3H, H6), 1.73 (s, 3H, CH₃), 2.49-2.65 (m, 2H, H3),4.23 (t, J=6.7 Hz, 1H, H9′), 4.40 (d, J=6.7 Hz, 2H, CH₂O), 5.11 (m, 1H,H4), 5.41 (bd, J=7.5 Hz, 1H, NH), 7.31 (t, J=7.3 Hz, 2H, H2′, 7′), 7.40(t, J=7.3 Hz, 2H, H3′, 6′), 7.58-7.66 (m, 2H, H1′, 8′), 7.76 (d, J=7.4Hz, 2H, H4′, 5′), 9.22 (bs, 1H, OH). ¹³C n.m.r. (100 MHz, CDCl₃): δ 18.1(C6), 26.0 (CH₃C═), 30.8 (C3), 47.3 (C9′), 53.8 (C2), 67.2 (CH₂O), 117.5(C4), 120.1 (C2′, 7′), 125.2 (C3′, 6′), 127.2 (Cr, 8′), 127.8 (C4′, 5′),136.9 (C5), 141.4, 143.9 (Arom C), 156.1 (CONH), 176.2 (C1). HRMS (ESI⁺,MeOH). Found: m/z 388.1522 (M+Na)⁺, C₂₂H₂₃NNaO₄ requires 388.1525. Theproduct later crystallised on standing to give a pale brown solid, m.p.109-111° C.

7.15 Pentapeptide Transformations 7.15.1 Linear:Fmoc-Hag-Ala-Trp-Arg-Hag-NH₂ 94

The procedure described in Section 73.2.2 was used for the attachment ofthe first amino acid, Fmoc-L-Hag-OH 96, to Rink amide resin. Quantitiesof the resin and coupling reagents HATU and NMM are presented in Table7.1. The first coupling reaction was shaken for 14 h.

TABLE 7.1 Quantities of Reagents used in the Synthesis of Peptide 94Reagent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 155 mg0.11 Fmoc-L-Hag-OH 110 mg 0.33 HATU 83.0 mg 0.22 NMM 71.8 μl 0.65

The procedure outlined in Section 7.3.2.2 was also utilised forsubsequent coupling reactions in the synthesis of the pentapeptide 94.Quantities of the coupling agents HATU and NMM remained constantthroughout the synthesis. The quantities of successive amino acids andtheir reaction durations are detailed in Table 7.2.

TABLE 7.1 Quantities of Amino Acids used in the Synthesis of Peptide 94Amino Acid Mass (mg) Mote (mmol) Reaction Time (h)* Fmoc-L-Arg(Pbf)-OH211 0.33 5 Fmoc-L-Trp(Boc)-OH 171 0.32 3 Fmoc-L-Ala-OH 102 0.33 4.5Fmoc-L-Hag-OH 110 0.33 20 *Note: Reaction times have not been optimised.

After the final amino acid coupling, a small aliquot of peptidyl-resinwas subjected to the TFA-mediated cleavage procedure (Section 7.3.3).Mass spectral analysis of the isolated residue confirmed formation ofthe pentapeptide 94. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 847.1 (M+H)⁺,C₄₅H₅₅N₁₀O₇ requires 847.4.

7.15.2 Linear: Fmoc-Crt-Ala-Trp-Arg-Crt-NH₂ 99

The procedure described in Section 7.3.2.2 was used for the attachmentof the first amino acid, Fmoc-L-Crt-OH 100, to Rink amide resin.Quantities of the resin and coupling reagents HATU and NMM are presentedin Table 7.3. The first coupling reaction was shaken for 3 h.

TABLE 7.3 Quantities of Reagents used in the Synthesis of Peptide 99Reagent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 110 mg0.08 Fmoc-L-Crt-OH 81.5 mg 0.23 HATU 58.6 mg 0.15 NMM 51.0 μl 0.46

The procedure outlined in Section 7.3.2.2 was also utilised forsubsequent coupling reactions in the synthesis of the pentapeptide 99.Quantities of the coupling agents HATU and NMM remained constantthroughout the synthesis. The quantities of successive amino acids andtheir reaction durations are detailed in Table 7.4.

TABLE 7.4 Quantities of Amino Acids used in the Synthesis of Peptide 99Amino Acid Mass (mg) Mole (mmol) Reaction Time (h)* Fmoc-L-Arg(Pbf)-OH150 0.23 20 Fmoc-L-Trp(Boc)-OH 122 0.23 4 Fmoc-L-Ala-OH 72.0 0.23 2Fmoc-L-Crt-OH 81.5 0.23 12 *Note: Reaction times have not beenoptimised.

After the final amino acid coupling, a small aliquot of peptidyl-resinwas subjected to the TFA-mediated cleavage procedure (Section 7.3.3).Mass spectral analysis of the isolated residue confirmed formation ofthe pentapeptide 99. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 875.2 (M+H)⁺,C₄₇H₅₉N₁₀O₇ requires 875.4.

7.15.3 Linear: Fmoc-Hag-Pro-Trp-Arg-Hag-NH₂ 97

The procedure described in Section 7.3.2.2 was used for the attachmentof the first amino acid, Fmoc-L-Hag-OH 96, to Rink amide resin.Quantities of the resin and coupling reagents HATU and NMM are presentedin Table 7.5. The first coupling reaction was shaken for 14 h.

TABLE 7.5 Quantities of Reagents used in the Synthesis of Peptide 97Reagent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 154 mg0.11 Fmoc-L-Hag-OH 109 mg 0.32 HATU 82 mg 0.22 NMM 71.4 μl 0.65

The procedure outlined in Section 7.3.2.2 was also utilised forsubsequent coupling reactions in the synthesis of the pentapeptide 97.Quantities of the coupling agents HATU and NMM remained constantthroughout the synthesis. The quantities of successive amino acids andtheir reaction durations are detailed in Table 7.6

TABLE 0.2 Quantities of Amino Acids used in the Synthesis of Peptide 97Amino Acid Mass (mg) Mole (mmol) Reaction Time (h)* Fmoc-L-Arg(Pbf)-OH210 0.32 5 Fmoc-L-Trp(Boc)-OH 170 0.32 3 Fmoc-L-Pro-OH 110 0.33 4.5Fmoc-L-Hag-OH 109 0.32 22 *Note: Reaction times have not been optimised.

After the final amino acid coupling, a small aliquot of peptidyl-resinwas subjected to the TFA-mediated cleavage procedure (Section 7.3.3).Mass spectral analysis of the isolated residue confirmed formation ofthe pentapeptide 97. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 873.2 (M+H)⁺,C₄₇H₅₇N₁₀O₇ requires 873.4; 895.1 (M+Na)⁺, C₄₇H₅₆N₁₀NaO₇ requires 895.4.

7.15.4 Unsaturated Cyclic: Fmoc-c[Hag-Ala-Trp-Arg-Hag]-NH₂ 95

Method A:

The resin-bound peptide 94a was subjected to the conventional RCMprocedure (Section 7.5.2) under the following conditions: Resin-peptide94a (20.0 mg, 14.0 μmol), DCM (3 mL), LiCl/DMF (0.4 M, 0.3 mL), Grubbs'catalyst (2.3 mg, 2.8 μmol, 20 mol %), 50° C., 41 h. At the end of thereaction period, a small aliquot of peptidyl-resin was subjected to theTFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysisof the isolated residue indicated recovery of the starting linearpeptide 94. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 847.2 (M+H)⁺ _(linear),C₄₅H₅₅N₁₀O₇.

Method B:

The resin-bound peptide 94a was subjected to the conventional RCMprocedure (Section 7.5.2) under the following conditions: Resin-peptide94a (37.0 mg, 25.9 μmol), DCM (3 mL), LiCl/DMF (0.4 M, 0.3 mL), 2^(nd)generation Grubbs' catalyst (4.4 mg, 5.2 μmol, 20 mol %), 50° C., 41 h.At the end of the reaction period, a small aliquot of peptidyl-resin wassubjected to the TFA-mediated cleavage procedure (Section 0). Massspectral analysis of the isolated residue confirmed the presence of bothcyclic 95 and linear 94 peptides. Mass spectrum (ESI⁺, MeCN/H₂O): m/z819.2 (M+H)⁺ _(cyclic), C₄₃H₅₁N₁₀O₇ requires 819.4; m/z 847.2 (M+H)⁺_(linear), C₄₅H₅₅N₁₀O₇.

Method C:

The resin-bound peptide 99a was subjected to the conventional RCMprocedure (Section 7.5.2) under the following conditions: Resin-peptide99a (32.8 mg, 23.0 μmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2^(nd)generation Grubbs' catalyst (4.0 mg, 4.7 μmol, 20 mol %), 50° C., 41 h,100% conversion into 95. At the end of the reaction period, a smallaliquot of peptidyl-resin was subjected to the TFA-mediated cleavageprocedure (Section 7.3.3). Mass spectral analysis of the isolatedresidue confirmed formation of the cyclic peptide 95.^(†) Mass spectrum(ESI⁺, MeCN/H₂O): m/z 819.2 (M+H)⁺ _(cyclic), C₄₃H₅₁N₁₀O₇ requires819.4. ^(†) RCM of the crotylglycine-containing peptide 99 leads to thesame unsaturated carbocycle 95 resulting from cyclisation of theallylglycine-containing sequence 94, i.e. Fmoc-c[Hag-Ala-Trp-Arg-Hag]-OHis identical to Fmoc-c[Crt-Ala-Trp-Arg-Crt]-OH.

7.15.5 Unsaturated Cyclic: Fmoc-c[Hag-Pro-Trp-Arg-Hag]-NH₂ 98

Method A:

The resin-bound peptide 97a was subjected to the conventional RCMprocedure (Section 7.5.2) under the following conditions: Resin-peptide97a (26.4 mg, 18.5 DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), Grubbs'catalyst (6.1 mg, 7.4 μmol, 20 mol %), 50° C., 41 h. At the end of thereaction period, a small aliquot of peptidyl-resin was subjected to theTFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysisof the isolated residue indicated recovery of the starting linearpeptide 97. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 873.2 (M+H)⁺ _(linear),C₄₇H₅₇N₁₀O₇.

Method B:

The resin-bound peptide 97a was subjected to the conventional RCMprocedure (Section 7.5.2) under the following conditions: Resin-peptide97a (36.0 mg, 25.2 μmol), DCM (3 mL), LiCl/DMF (0.4 M, 0.3 mL), 2^(nd)generation Grubbs' catalyst (4.4 mg, 5.2 μmol, 20 mol %), 50° C., 41 h,100% conversion into 98. At the end of the reaction period, a smallaliquot of peptidyl-resin was subjected to the TFA-mediated cleavageprocedure (Section 7.3.3). Mass spectral analysis of the isolatedresidue confirmed formation of the cyclic peptide 98. Mass spectrum(ESI⁺, MeCN/H₂O): m/z 845.1 (M+H)⁺, C₄₅H₅₃N₁₀O₇ requires 845.4; 867.1(M+Na)⁺,C₄₅H₅₂N₁₀NaO₇ requires 867.4.

7.15.6 Linear: Fmoc-Hag-Pro-Pre-Arg-Hag-OH 102

The procedure outlined in Section 7.3.2.1 was used for the attachment ofthe first amino acid, Fmoc-Hag-OH 96, to Wang resin. Quantities of theresin and coupling reagents are presented in Table 7.7. The firstcoupling reaction was shaken for 14 h.

TABLE 7.7 Quantities of Reagents used in the Synthesis of Peptide 102Reagent Mass (mg) or Volume (μl) Mole (mmol) Wang Resin 212 mg 0.19Fmoc-L-Hag-OH 195 mg 0.58 DIC 90.6 μl 0.58 DMAP 7.1 mg 0.06

The procedure outlined in Section 7.3.2.1 was also utilised forsubsequent coupling reactions in the synthesis of the pentapeptide 102.Quantities of the coupling reagents HATU and NMM are tabulated (Table7.8) and remained constant throughout the synthesis. The quantities ofsuccessive amino acids and their reaction durations are detailed inTable 7.9.

TABLE 7.8 Quantities of Coupling Reagents used in the Synthesis ofPeptide 102 Coupling Reagent Mass (mg) or Volume (mL) Mole (mmol) HATU147 mg 0.39 NMM 128 μl 1.16

TABLE 7.9 Quantities of Amino Acids used in the Synthesis of Peptide 102Amino Acid Mass (mg) Mole (mmol) Reaction Time (h) Fmoc-L-Arg(Pbf)-OH376 0.58 2 Fmoc-L-Pre-OH 211 0.58 3 Fmoc-L-Pro-OH 196 0.58 6Fmoc-L-Hag-OH 195 0.58  2 (1) *Note: Reaction times have not beenoptimised.

After the final amino acid coupling, a small aliquot of peptidyl-resinwas subjected to the TFA-mediated cleavage procedure (Section 7.3.3).Mass spectral analysis of the isolated residue confirmed formation ofthe pentapeptide 102. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 813.6 (M+H)⁺,C₄₃H₅₇N₈O₈ requires 813.4; m/z 831.5 (M+H₂O+H)⁺ ₁₀₃, C₄₃H₅₉N₈O₉ requires831.4; m/z 927.6 (M+TFA+H)⁺, C₄₅H₅₈F₃N₈O₁₀ requires 927.4.

The pentapeptide 102 was also synthesised on Wang resin (590 mg) withreduced loading (0.3 mmol g⁻¹) using the procedured described above. Therelative quantities of the Fmoc-amino acids and coupling agents remainedconstant throughout the synthesis: Wang resin:DIC:DMAP:Fmoc-aminoacid:HATU:NMM, 1:3:0.3:3:2:6 equiv.

7.15.7 Unsaturated Cyclic: Fmoc-c[Hag-Pro-Pre-Arg-Hag]-OH 104

The resin-bound peptide 102a was subjected to the conventional RCMprocedure (Section 7.5.2) under the following conditions: Resin-peptide102a (70.0 mg, 63.7 μmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2^(nd)generation Grubbs' catalyst (21.6 mg, 25.4 μmol, 40 mol %), 50° C., 42h, 100% conversion into 104. At the end of the reaction period, a smallaliquot of peptidyl-resin was subjected to the TFA-mediated cleavageprocedure (Section 7.3.3). Mass spectral analysis of the isolatedresidue confirmed formation of the cyclic peptide 104. Mass spectrum(ESI⁺, MeCN/H₂O): m/z 785.4 (M+H)⁺, C₄₁H₅₃N₈O₈ requires 785.4; m/z 803.3(M+H₂O+H)⁺, C₄₁H₅₅N₈O₉ requires 803.4; m/z 899.4 (M+TFA+H)⁺,C₄₃H₅₄F₃N₈O₁₀ requires 899.4.

The resin-bound peptide 102a (synthesised on reduced loading Wang resin)was subjected to the conventional RCM procedure (Section 7.5.2) underthe following conditions: Resin-peptide 102a (97.0 mg, 29.1 μmol), DCM(5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2^(nd) generation Grubbs' catalyst(2.5 mg, 2.9 μmol, 10 mol %), 50° C., 42 h, 100% conversion into 104.Mass spectral data of the isolated residue confirmed formation of thecyclic peptide 104 and were in agreement with those reported above.

7.15.8 Saturated Cyclic: Fmoc-c [Hag-Pro-Pre-Arg-Hag]-OH 105

The resin-bound peptide 104a was subjected to the general Wilkinson'shydrogenation procedure (Section 7.4.4) under the following conditions:Resin-peptide 104a (350 mg, 0.32 mmol), DCM:MeOH (9:1, 8 mL),Wilkinson's catalyst, 80 psi H₂, 22° C., 22 h, 100% conversion into 105.At the end of the reaction period, a small aliquot of peptidyl-resin wassubjected to the TFA-mediated cleavage procedure (Section 7.3.3). Massspectral analysis of the isolated residue confirmed formation of thesaturated cyclic pentapeptide 105. Mass spectrum (ESI⁺, MeCN/H₂O): m/z787.2 (M+H)⁺, C₄₁H₅₅N₈O₈ requires 787.4; m/z 805.2 (M+H₂O+H)⁺,C₄₁H₅₇N₈O₉ requires 803.4; m/z 901.3 (M+TFA+H)⁺, C₄₃H₅₆F₃N₈O₁₀ requires901.4.

7.15.9 Olefin Activation: Saturated Cyclic:Fmoc-c[Hag-Pro-Crt-Arg-Hag]-OH 106

The resin-bound peptide 105a was subjected to the general crossmetathesis procedure (Section 7.5.4) with cis-2-butene under thefollowing conditions: Resin-peptide 105a (212 mg, 0.19 mmol), ICM (8mL), 2^(nd) generation Grubbs' catalyst (82 mg, 9.7 μmol, 50 mol %),cis-2-butene (15 psi), 50° C., 42 h. At the end of the reaction period,a small aliquot of peptidyl-resin was subjected to the TFA-mediatedcleavage procedure (Section 7.3.3). Mass spectral analysis of theisolated residue indicated the presence of the starting peptide 105 andthe desired butenolysis product 106. The recovered resin-peptide wassubjected to the same butenolysis conditions in order to drive thereaction to completion. After 42 h, a small aliquot of peptidyl-resinwas subjected to the TFA-mediated cleavage procedure (Section 7.3.3).Mass spectral analysis of the isolated residue confirmed quantitativeconversion to the activated peptide 106. Mass spectrum (ESI⁺, MeCN/H₂O):m/z 773.2 (M+H)⁺, C₄₀H₅₃N₈O₈ requires 773.4.

7.15.10 Cross Metathesis of Activated Olefin: SaturatedCyclicFmoc-c[Hag-Pro-Sub-Arg-Hag]-OH 107

The resin-bound peptide 106a was subjected to the generalmicrowave-accelerated cross metathesis procedure (Section 7.5.3) underthe following conditions: Resin-peptide 106a (20.0 mg, 18.0 μmol), DCM(4 mL), LiCl/DMF (0.4 M, 0.4 mL), 2^(nd) generation Grubbs' catalyst(6.2 mg, 7.3 μmol, 40 mol %), (2S)-methyl 2-N-acetylaminohex-4-enoate 81(70.0 mg, 0.38 mmol), 100° C., 2 h. At the end of the reaction period, asmall aliquot of peptidyl-resin was subjected to the TFA-mediatedcleavage procedure (Section 7.3.3). Mass spectral analysis of theisolated residue confirmed formation of the cross metathesis product107. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 902.4 (M+H)⁺, C₄₅H₆₀N₉O₁₁requires 902.4.

7.15.11 Wilkinson's Hydrogenation of Saturated Cyclic 107:Fmoc-c[Hag-Pro-sat(Sub)-Arg-Hag]-OH 108

The resin-bound peptide 107a was subjected to the general Wilkinson'shydrogenation procedure (Section 7.4.4) under the following conditions:Resin-peptide 107a (15.0 mg, 13.5 μmol), DCM:MeOH (9:1, 5 mL),Wilkinson's catalyst, 80 psi H₂, 22° C., 22 h. At the end of thereaction period, a small aliquot of peptidyl-resin was subjected to theTFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysisof the isolated residue confirmed formation of the reduced cyclicpentapeptide 108. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 904.4 (M+H)⁺,C₄₅H₆₂N₉O₁₁ requires 904.5.

7.15.12 Olefin Activation: Synthesis of Fmoc-Gly(CH₂CH═CHCH₂OAc)-Phe-OH145

The resin-bound peptide Fmoc-Pre-Phe-Wang 144 was subjected to themicrowave-assisted cross metathesis procedure (Section 7.5.3) withcis-1,4-diacetoxy-2-butene 141 under the following conditions: Resin(Wang)-peptide 144 (180 mg, 0.09 mmol), DCM (10 mL), 2^(nd) generationGrubbs' catalyst (16 mg, 20 mol %), cis-1,4-diacetoxy-2-butene (96 mg,0.56 mmol, 15 psi), 100° C., 1 h. At the end of the reaction period, thepeptidyl-resin was subjected to the TFA-mediated cleavage procedure(Section 7.3.3). Mass spectral analysis of the isolated residueindicated the presence of the desired dipeptide product 145 and nostarting material. Mass spectral analysis of the isolated residueconfirmed quantitative conversion to the activated peptide 145. Massspectrum (ESI⁺, CH₃OH): m/z 579.0 (M+Na) C₃₂H₃₂N₂O₇Na.

7.16 [2,8]-Dicarba-[3,12]-Cystino Conotoxin Transformations 7.16.1Linear [2,8]-Hag-[3,12]-Cys Conotoxin ImI:Fmoc-Gly-Hag-Cys-Ser-Asp-Pro-Arg-Hag-Ala-Trp-Arg-Cys-NH₂ 112

The procedure described in Section 7.3.2 was used for the attachment ofthe first amino acid, Fmoc-L-Cys(Trt)-OH, to Rink amide resin.Quantities of the resin and coupling reagents HATU and NMM are presentedin Table 7.10. The first coupling reaction was shaken for 14 h.

TABLE 7.10 Quantities of Reagents used in the Synthesis of Peptide 112Reagent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 740 mg0.39 Fmoc-L-Cys(Trt)-OH 676 mg 1.15 HATU 293 mg 0.77 NMM 255 μl 2.31

The procedure outlined in Section 7.3.2.2 was also utilised forsubsequent coupling reactions in the synthesis of the dodecapeptide 112.Quantities of the coupling agents HATU and NMM remained constantthroughout the synthesis. The quantities of successive amino acids andtheir reaction durations are detailed in Table 7.11.

TABLE 7.11 Quantities of Amino Acids used in the Synthesis of Peptide112 Amino Acid Mass (mg) Mole (mmol) Reaction Time (h)*Fmoc-L-Arg(Pbf)-OH 749 1.15 2.5 Fmoc-L-Trp(Boc)-OH 608 1.15 2.5Fmoc-L-Ala-OH 360 1.16 14 Fmoc-L-Hag-OH 390 1.16 2.5 Fmoc-L-Arg(Pbf)-OH750 1.16 2.5 Fmoc-L-Pro-OH 390 1.16 1 Fmoc-L-Asp(^(t)Bu)—OH 475 1.15 14Fmoc-L-Ser(^(t)Bu)—OH 443 1.16 2.5 Fmoc-L-Cys(Trt)-OH 676 1.15 2.5Fmoc-L-Hag-OH 390 1.16 2.5 Fmoc-L-Gly-OH 343 1.15 14 *Note: Reactiontimes have not been optimised.

After the final amino acid coupling, a small aliquot of peptidyl-resinwas subjected to the TFA-mediated cleavage procedure (Section 7.3.3).Mass spectral analysis of the isolated residue confirmed formation ofthe dodecapeptide 112. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 783.5[½(M+2H)]⁺, ½(C₇₁H₉₈N₂₀O₁₇S₂) requires 783.3; m/z 1565.7 (M+H)⁺,C₇₁H₉₇N₂₀O₁₇S₂ requires 1565.7. LC-MS (Luna C8 RP-column, 10-60% MeOH,0.1% formic acid): t_(R)=8.86 min.

7.16.2 [2,8]-Dicarba-[3,12]-Cys Conotoxin ImI:Fmoc-Gly-c[Hag-Cys-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Cys-NH₂ 114

The resin-bound peptide was subjected to the generalmicrowave-accelerated RCM procedure (Section 7.5.3) under the followingconditions: Resin-peptide 112a (158 mg, 82.2 μmol), DCM (3 mL), LiCl/DMF(0.4 M, 0.3 mL), 2^(nd) generation Grubbs' catalyst (7.0 mg, 8.2 μmol,10 mol %), 100° C., 1 h, 100% conversion into 114. At the end of thereaction period, a small aliquot of peptidyl-resin was subjected to theTFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysisof the isolated residue confirmed formation of the cyclic peptide 114.Mass spectrum (ESI⁺, MeCN/H₂O): m/z 769.4 [½(M+2H)]⁺, ½(C₆₉H₉₄N₂₀O₁₇S₂)requires 769.3; m/z 1537.7 (M+H)⁺, C₆₉H₉₃N₂₀O₁₇S₂ requires 1537.6. LC-MS(Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): t_(R)=8.59 min.

An analogous microwave-accelerated RCM reaction using 5 mol % 2^(nd)generation Grubbs' catalyst was performed: Resin-peptide 112a (80.2 mg,42 μmol), DCM (3 mL), LiCl/DMF (0.4 M, 0.3 mL), 2^(nd) generationGrubbs' catalyst (7.0 mg, 2.1 μmol, 5 mol %), 100° C., 2 h, 100%conversion into 114. Mass spectral data were in agreement with thosereported above.

7.16.3 [2,8]-Dicarba-[3,12]-Cystino Conotoxin ImI:NH₂-Gly-c[Hag-Cys-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Cys)-NH₂ 118

The Rink-amide bound peptide 114a (100 mg, 52.0 μmol) was swollen withDCM (3×1 min, 1×30 min) and DMF (3×1 min, 1×30 min) and deprotected with20% piperidine/DMF (1×1 min, 2×20 min). The resin was then washed withDMF (5×1 min), DCM (3×1 min), MeOH (3×1 min) and dried on the SPPSmanifold for 1 h. The Fmoc-deprotected peptidyl-resin (47.0 mg, 24.4μmol) was subjected to the TFA-mediated cleavage procedure (Section 0).The residue was then lyophilised for 18 h to give the fully deprotectedcarbocyclic peptide 116 as a colourless solid (20.0 mg, 15.2 μmol). Massspectrum (ESI⁺, MeCN/H₂O): m/z 658.4 [½(M+2H)]⁺, ½(C₅₄H₈₄N₂₀O₁₅S₂)requires 658.3; m/z 1315.6 (M+H)⁺, C₅₄H₈₃N₂₀O₁₅S₂ requires 1315.6. LC-MS(Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): t_(R)=5.63 min

A sample of lyophilised peptide (10.1 mg, 7.7 μmol) was dissolved in anaqueous solution of (NH₄)₂CO₃ (0.1 M, 80 mL) containing 5% DMSO (4 mL).The reaction was stirred at room temperature and monitored by theEllman's test (Section 7.3.4). After 3 d, the reaction mixture waslyophilised and mass spectral analysis of the isolated residue confirmedformation of the cystine-oxidised peptide 118. The peptide was purifiedby RP-HPLC (Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid) and theunsaturated [2,8]-dicarba-[3,12]-cystino conotoxin hybrid 118 wasisolated as a colourless solid (1.8 mg, 5%) in >99% purity. Massspectrum (ESI⁺, MeCN/H₂O): m/z 657.4 [½(M+2H)]⁺, ½(C₅₄H₈₂N₂₀O₁₅S₂)requires 657.3; m/z 668.3 [½(M+H+Na)]⁺, ½(C54H₈₁N₂₀NaO₁₅S₂) requires668.3; m/z 1313.5 (M+H)⁺, C₅₄H₈₁N₂₀O₁₅S₂ requires 1313.6. LC-MS (Luna C8RP-column, 10-60% MeOH, 0.1% formic acid): t_(R)=5.50 min.

7.16.4 [2,8]-Saturated Dicarba-[3,12]-Cystino Conotoxin ImI:NH₂-Gly-c[Hag-Cys-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Cys-NH₂ 122

The resin-bound peptide 114a was subjected to the general Wilkinson'shydrogenation procedure (Section 7.4.4) under the following conditions:Resin-peptide 114a (285 mg, 0.15 mmol), DCM:MeOH (9:1, 5 mL),Wilkinson's catalyst, 80 psi H₂, 22° C., 22 h. At the end of thereaction period, a small aliquot of peptidyl-resin was Fmoc-deprotected(20% piperidine/DMF, 1×1 min, 2×10 min) and washed with DMF (5×1 min),DCM (5×1 min), MeOH (5×1 min) and dried on the SPPS manifold for 1 h.The Fmoc-deprotected peptidyl-resin was then subjected to theTFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysisof the isolated residue indicated the presence of a mixture of thecystine-oxidised 122 and reduced 120 form of the saturated product. Massspectrum (ESI⁺, MeCN/H₂O): m/z 658.6 [½(M+2H)]⁺ _(oxidised),½(C₅₄H₈₄N₂₀O₁₅S₂) requires 658.3; m/z 1315.7 (M+H)⁺ _(oxidised),C₅₄H₈₃N₂₀O₁₅S₂ requires 1315.6; m/z 659.4 [½(M+2H)]⁺ _(reduced),½(C₅₄H₈₆N₂₀O₁₅S₂) requires 659.3; m/z 1317.8 (M+H)⁺ _(reduced),C₅₄H₈₅N₂₀O₁₅S₂ requires 1317.6. LC-MS (Luna C8 RP-column, 10-60% MeOH,0.1% formic acid): t_(R) (122)=6.01 min.

7.17 [3,12]-Dicarba-[2,8]-Cystino Conotoxin Transformations 7.17.1Linear [2,8]-Cys-[3,12]-Hag Conotoxin ImI:Fmoc-Gly-Cys-Hag-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Hag-NH₂ 113

The procedure described in Section 7.3.2.2 was used for the attachmentof the first amino acid, Fmoc-L-Hag-OH 96, to Rink amide resin.Quantities of the resin and coupling reagents HATU and NMM are presentedin Table 7.12. The first coupling reaction was shaken for 14 h.

TABLE 7.12 Quantities of Reagents used in the Synthesis of Peptide 113Reagent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 730 mg0.38 Fmoc-L-Hag-OH 384 mg 1.14 HATU 289 mg 0.76 NMM 250 μl 2.27

The procedure outlined in Section 7.3.2.2 was also utilised forsubsequent coupling reactions in the synthesis of the dodecapeptide 113.Quantities of the coupling agents HATU and NMM remained constantthroughout the synthesis. The quantities of successive amino acids andtheir reaction durations are detailed in Table 7.13.

TABLE 7.13 Quantities of Amino Acids used in the Synthesis of Peptide113 Amino Acid Mass (mg) mole (mmol) Reaction Time (h)*Fmoc-L-Arg(Pbf)-OH 739 1.14 2.5 Fmoc-L-Trp(Boc)-OH 600 1.14 2.5Fmoc-L-Ala-OH 355 1.14 14 Fmoc-L-Cys(Trt)-OH 667 1.14 2.5Fmoc-L-Arg(Pbf)-OH 740 1.14 2.5 Fmoc-L-Pro-OH 385 1.14 1Fmoc-L-Asp(^(t)Bu)—OH 470 1.14 14 Fmoc-L-Ser(^(t)Bu)—OH 437 1.14 2.5Fmoc-L-Hag-OH 385 1.14 2.5 Fmoc-L-Cys(Trt)-OH 667 1.14 2.5 Fmoc-L-Gly-OH340 1.14 14 *Note: Reaction times have not been optimised.

After the final amino acid coupling, a small aliquot of peptidyl-resinwas subjected to the TFA-mediated cleavage procedure (Section 7.3.3).Mass spectral analysis of the isolated residue confirmed formation ofthe dodecapeptide 113. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 783.5[½(M+2H)]⁺, ½(C₇₁H₉₈N₂₀O₁₇S₂) requires 783.3; m/z 1565.7 (M+H)⁺,C₇₁H₉₇N₂₀O₁₇S₂ requires 1565.7. LC-MS (Luna C8 RP-column, 10-60% MeOH,0.1% formic acid): t_(R)=9.13 min.

7.17.2 [2,8]-Cys-[3,12]-Dicarba Conotoxin ImI:Fmoc-Gly-Cys-c[Hag-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Hag]-NH₂ 115

The resin-bound peptide 113a was subjected to the generalmicrowave-accelerated RCM procedure (Section 7.5.3) under the followingconditions: Resin-peptide 113a (840 mg, 0.44 mmol), DCM (5 mL), LiCl/DMF(0.4 M, 0.5 mL), 2^(nd) generation Grubbs' catalyst (74.3 mg, 87.5 μmol,20 mol %), 100° C., 1 h, 100% conversion into 115. At the end of thereaction period, a small aliquot of peptidyl-resin was subjected to theTFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysisof the isolated residue confirmed formation of the cyclic peptide 115.Mass spectrum (ESI⁺, MeCN/H₂O): m/z 769.4 [½(M+2H)]⁺, ½(C₆₉H₉₄N₂₀O₁₇S₂)requires 769.3; m/z 1537.7 (M+H)⁺, C₆₉H₉₃N₂₀O₁₇S₂ requires 1537.6. LC-MS(Luna C8 RP-column, 10-60% MeOH, 0.1% formic acid): t_(R)=8.99 min.

7.17.3 [2,8]-Cystino-[3,12]-Dicarba Conotoxin ImI:NH₂-Gly-Cys-c[Hag-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Hag]-NH₂ 119

The resin-bound peptide 115a (100 mg, 52.0 μmol) was swollen with DCM(3×1 min, 1×30 min) and DMF (3×1 min, 1×30 min) and deprotected with 20%piperidine/DMF (1×1 min, 2×20 min). The resin was then washed with DMF(5×1 min), DCM (3×1 min), MeOH (3×1 min) and dried on the SPPS manifoldfor 1 h. The Fmoc-deprotected peptidyl-resin (61.7 mg, 32.1 μmol) wassubjected to the TFA-mediated cleavage procedure (Section 7.3.3). Theresidue was then lyophilised for 18 h to give the fully deprotectedcarbocyclic peptide 117 as a colourless solid (15.1 mg, 11.5 μmol). Massspectrum (ESI⁺, MeCN/H₂O): m/z 658.4 [½(M+2H)]⁺, ½(C₅₄H₈₄N₂₀O₁₅S₂)requires 658.3; m/z 669.4 [½(M+H+Na)]⁺, ½(C₅₄H₈₄N₂₀O₁₅S₂) requires669.4; m/z 1315.6 (M+H)⁺, C₅₄H₈₃N₂₀O₁₅S₂ requires 1315.6. LC-MS (Luna C8RP-column, 10-60% MeOH, 0.1% formic acid): t_(R)=6.62 min.

A sample of lyophilised peptide (11.2 mg, 8.5 μmol) was dissolved in anaqueous solution of (NH₄)₂CO₃ (0.1 M, 80 mL) containing 5% DMSO (4 mL).The reaction was stirred at room temperature and monitored by theEllman's test. After 3 d, the reaction mixture was lyophilised and massspectral analysis of the isolated residue confirmed formation of thecystine-oxidised peptide 119. The peptide was purified by RP-HPLC (LunaC8 RP-column, 10-60% MeOH, 0.1% formic acid) and the unsaturated[2,8]-cystino-[3,12]-dicarba conotoxin hybrid 119 was isolated as acolourless solid (2.3 mg, 5%) in >99% purity. Mass spectrum (ESI⁺,MeCN/H₂O): m/z 657.3 [½(M+2H)]⁺, ½(C₅₄H₈₂N₂₀O₁₅S₂) requires 657.3; m/z668.3 [½(M+H+Na)]⁺, ½(C₅₄H₈₁N₂₀NaO₁₅S₂) requires 668.3; m/z 1313.6(M+H)⁺, C₅₄H₃₁N₂₀O₁₅S₂ requires 1313.6. LC-MS (Luna C8 RP-column, 10-60%MeOH, 0.1% formic acid): t_(R)=4.46 min.

7.17.4 [2,8]-Cystino[3,12]-Saturated Dicarba Conotoxin ImI:NH₂-Gly-Cys-c[Hag-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Hag]-NH₂ 123

The resin-bound peptide 119a was subjected to the general Wilkinson'shydrogenation procedure (Section 7.4.4) under the following conditions:Resin-peptide 119a (320 mg, 0.17 mmol), DCM:MeOH (9:1, 5 mL),Wilkinson's catalyst, 80 psi H₂, 22° C., 22 h. At the end of thereaction period, a small aliquot of peptidyl-resin was Fmoc-deprotected(20% piperidine/DMF, 1×1 min, 2×10 min) and washed with DMF (5×1 min),DCM (5×1 min), MeOH (5×1 min) and dried on the SPPS manifold for 1 h.The Fmoc-deprotected peptidyl-resin was then subjected to theTFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysisof the isolated residue indicated the presence of a mixture of thecystine-oxidised 123 and reduced 121 form of the saturated product. Massspectrum (ESI⁺, MeCN/H₂O): m/z 658.5 [½(M+²H)]⁺ _(oxidised),½(C₅₄H₈₄N₂₀O₁₅S₂) requires 658.3; m/z 1315.7 (M+H)⁺ _(oxidised),C₅₄H₈₃N₂₀O₁₅S₂ requires 1315.6; m/z 659.3 [½(M+2H)]⁺ _(reduced),½(C₅₄H₈₆N₂₀O₁₅S₂) requires 659.3; m/z 1317.6 (M+H)⁺ _(reduced),C₅₄H₈₅N₂₀O₁₅S₂ requires 1317.6. LC-MS (Luna C8 RP-column, 10-60% MeOH,0.1% formic acid): t_(R) (123)=7.02 min.

7.17.5 Linear [2,8]-Hag-[3,16]-Cystino Conotoxin Vc1.1 (ACV1)

Gly-Hag-Cys-Ser-Asp-Pro-Arg-Hag-Asn-Tyr-Asp-His-Pro-Glu-Ile-Cys-NH₂

The procedure described in Section 7.3.5 was used for the synthesis ofVc1.1 on Rink Amide resin (loading 0.52 mmol/g). Quantities of theresin, coupling reagents and amino acids are tabulated below:

Mole Quantity (mmol)/ Compound (mL/g) Volume Conc (M) Cycle Name RinkAmide 0.481 g 5 mL DMF 0.25 mmol — DIPEA 7.7 mL 22 mL NMP 2M — HBTU6.827 g 36 mL DMF 0.45M — HOBt 2.432 g Fmoc-Arg- 0.389 3 mL DMF 0.2MB0.25-Single (ext.) OH Fmoc-Asn- 0.358 3 mL DMF 0.2M B0.25-Single (ext.)OH Fmoc-Asp- 0.494 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Cys- 0.7036 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Glu- 0.255 3 mL DMF 0.2MB0.25-Single (ext.) OH Fmoc-Gly- 0.178 3 mL DMF 0.2M B0.25-Single (ext.)OH Fmoc-His- 0.372 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Ile- 0.2123 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Pro- 0.405 6 mL DMF 0.2MB0.25-Single (ext.) OH Fmoc-Ser- 0.230 3 mL DMF 0.2M B0.25-Single (ext.)OH Fmoc-Hag- 0.405 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Tyr- 0.2763 mL DMF 0.2M B0.25-Single (ext.) OH

Resin washings and deprotection cycles were performed as described inSection 7.3.5. The amino acid, activator and activator base solutionswere added to the resin, followed by the “B.01 Extended Coupling” cycle.The peptidyl-resin was exposed to a temperature of 75° C. with no power(0 watts) for 2 min, then at a temperature of 75° C., power at 25 wattsfor 10 min. The peptidyl-resin was then washed with DMF (3×10 mL).

Following the final amino acid coupling, a small aliquot of the resinbound peptide was cleaved as described in Section 7.3.3 for mass specanalysis. Mass spectrum (ESI⁺, MeOH/H₂O): m/z 592.8 (M+3H/3), m/z 1011.1(M+2H/2), m/z 1039.5 ((M+tBu)+3H/3).

7.17.6 [2,8]-Unsaturated-[3,16]-Cystino Conotoxin Vc1.1 (ACV1)

The resin bound linear peptide was subjected to microwave RCM procedureoutlined in Section 7.5.3. Peptidyl-resin (0.4810 mg, 0.25 mmol) and2^(nd) generation Grubb's catalyst (42.4 mg, 0.05 mmol) was weightedinto a glass vial loaded with stirrer bar. In a drybox, DCM (5 mL) andLiCl/DMF (0.2 mL) were added and the vial was sealed. The reactionvessel was placed in the microwave for 1 hr at 100 C. A small aliquot ofthe resin bound peptide was subjected to TFA cleavage and analysed bymass spectroscopy. Mass spectrum (ESI⁺, MeOH/H₂O): m/z 997.2 (M+2H/2),m/z 1011.0 (SM+2H/2). The same procedure was followed for ring closureof linear α-RgIA.

7.17.7 Linear [2,8]-Hag-[3,12]-Cystino Conotoxin α-RgIA from Conusregius

Gly-Hag-Cys-Ser-Asp-Pro-Arg-Hag-Arg-Tyr-Arg-Cys-Arg-N H₂

The procedure described in Section 7.3.5 was used for the synthesis ofRgIA on Rink Amide resin (loading 0.52 mmol/g). Quantities of the resin,coupling reagents and amino acids are tabulated in the Table below:

Mole Quantity (mmol)/ Compound (mL/g) Volume Conc (M) Cycle Name RinkAmide 0.192 g 5 mL DMF 0.10 mmol — DIPEA  3.8 mL 11 mL 2M — NMP HBTU2.655 g 18 mL 0.45M — HOBt 0.946 g DMF Fmoc-Arg- 1.427 g 11 mL 0.2MB0.25-Single (ext.) OH DMF Fmoc-Asp- 0.247 g 3 mL DMF 0.2M B0.25-Single(ext.) OH Fmoc-Cys- 0.703 g 6 mL DMF 0.2M B0.25-Single (cys/ OH hisext.) Fmoc-Gly- 0.178 g 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Pro-0.202 g 3 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Ser- 0.230 g 3 mL DMF0.2M B0.25-Single (ext.) OH Fmoc-Hag- 0.405 g 6 mL DMF 0.2M B0.25-Single(ext.) OH Fmoc-Tyr- 0.276 g 3 mL DMF 0.2M B0.25-Single (ext.) OH

Resin washings and deprotection cycles were performed as described inSection 7.3.5. The amino acid, activator and activator base solutionswere added to the resin, followed by the “B.01 Extended Coupling” cycle.The peptidyl-resin was exposed to a temperature of 75° C. with no power(0 watts) for 2 min, then at a temperature of 75° C., power at 25 wattsfor 10 min. The peptidyl-resin was then washed with DMF (3×10 mL).However cysteine residues in RgIA have been known to be susceptible toracemisation at 75° C., therefore a different cycle was used for thecoupling of this amino acid. Following the deprotection cycles, “B.01Single Cys/His Extended” coupling cycle was included in the method forthe coupling of cysteine. This involves exposure to a temperature of 50°C. with no power (0 watts) for 2 min, then at a temp of 50° C., power at25 watts for 10 min. The peptidyl resin was then washed with DMF (3×10mL).

Following the final amino acid coupling, a small aliquot of the resinbound peptide was cleaved as described in Section 7.3.3 for mass specanalysis. Mass spectrum (ESI⁺, MeOH/H₂O): m/z 595.5 (M+3H/3), m/z 614.3((M+tBu)+2H/2), m/z 892.6 (M+2H/2).

7.18 [2,8][3,12]-Dicarba Conotoxin Transformations 7.18.1 Linear[2,8]-Hag-[3,12]-Pre Conotoxin ImI:Fmoc-Gly-Hag-Pre-Ser-Asp-Pro-Arg-Hag-Ala-Trp-Arg-Pre-NH₂ 127

The procedure described in Section 7.3.2.2 was used for the attachmentof the first amino acid, Fmoc-L-Pre-OH 92, to Rink amide resin.Quantities of the resin and coupling reagents HATU and NMM are presentedin Table 7.14. The first coupling reaction was shaken for 4 h.

TABLE 7.14 Quantities of Reagents used in the Synthesis of Peptide 127Regent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 610 mg 0.32Fmoc-L-Pre-OH 350 0.96 HATU 241 mg 0.63 NMM 210 μl 1.91

The procedure outlined in Section 7.3.2.2 was also utilised forsubsequent coupling reactions in the synthesis of the dodecapeptide 127.The quantities of successive amino acids and their reaction durationsare detailed in Table 7.15.

TABLE 7.15 Quantities of Amino Acids used in the Synthesis of Peptide127 Amino Acid Mass (mg) Mole (mmol) Reaction Time (h)*Fmoc-L-Arg(Pbf)-OH 617 0.95 12 Fmoc-L-Trp(Boc)-OH 502 0.95 2.5Fmoc-L-Ala-OH 297 0.95 2.5 Fmoc-L-Hag-OH 321 0.95 2.5 Fmoc-L-Arg(Pbf)-OH617 0.95 14 Fmoc-L-Pro-OH 321 0.95 4 Fmoc-L-Asp(^(t)Bu)—OH 392 0.95 2.5Fmoc-L-Ser(^(t)Bu)—OH 365 0.95 2.5 Fmoc-L-Pre-OH 350 0.96 12Fmoc-L-Hag-OH 322 0.95 2.5 Fmoc-L-Gly-OH 285 0.96 2.5 (1) *Note:Reaction times have not been optimised.After the final amino acid coupling, a small aliquot of peptidyl-resinwas subjected to the TFA-mediated cleavage procedure (Section 7.3.3).Mass spectral analysis of the isolated residue confirmed fonnation ofthe dodecapeptide 127. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 805.6[½(M+2H)]⁺, ½(C₇₉H₁₁₀N₂₀O₁₇) requires 805.4; m/z 814.6 [½(M+H₂O+2H)]⁺,½(C₇₉H₁₁₂N₂₀O₁₈) requires 814.4.

7.18.2 [2,8]-Dicarba-[3,12]-Pre Conotoxin ImI:Fmoc-Gly-c[Hag-Pre-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Pre-NH₂ 129

Method A:

The Rink amide-bound peptide 127a was subjected to the conventional RCMprocedure (Section 7.5.2) under the following conditions: Resin-peptide127a (165 mg, 0.12 mmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2^(nd)generation Grubbs' catalyst (39.9 mg, 47.0 μmol, 40 mol %), 50° C., 40h, 100% conversion into 129. At the end of the reaction period, a smallaliquot of peptidyl-resin was subjected to the TFA-mediated cleavageprocedure (Section 7.3.3). Mass spectral analysis of the isolatedresidue confirmed formation of the cyclic peptide 129. Mass spectrum(ESI⁺, MeCN/H₂O): m/z 791.4 [½(M+2H)]⁺, ½(C₇₇H₁₀₆N₂₀O₁₇) requires 791.4.m/z 800.5 [½(M+H₂O+2H)]⁺, ½(C₇₇H₁₀₈N₂₀O₁₈) requires 800.4.

Method B:

The Rink amide-bound peptide 127a was subjected to the generalmicrowave-accelerated RCM procedure (Section 7.5.3) under the followingconditions: Resin-peptide 127a (127 mg, 66.0 μmol), DCM (5 mL), LiCl/DMF(0.4 M, 0.5 mL), 2^(nd) generation Grubbs' catalyst (5.6 mg, 6.6 μmol,10 mol %), 100° C., 1 h, 100% conversion into 129. At the end of thereaction period, a small aliquot of peptidyl-resin was subjected to theTFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysisof the isolated residue confirmed formation of the cyclic peptide 129.Mass spectrum (ESI⁺, MeCN/H₂O): m/z 791.5 [½(M+2H)]⁺, ½(C₇₇H₁₀₆N₂₀O₁₇)requires 791.4.

7.18.3 [2,8]-Saturated Dicarba-[3,12]-Pre Conotoxin ImI:Fmoc-Gly-c[Hag-Pre-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Pre-NH₂ 133

The resin-bound peptide 129a was subjected to the general Wilkinson'shydrogenation procedure (Section 7.4.4) under the following conditions:Resin-peptide 129a (130 mg, 91.0 μmol), DCM:MeOH (9:1, 6.5 mL),Wilkinson's catalyst, 80 psi H₂, 22° C., 24 h. At the end of thereaction period, a small aliquot of peptidyl-resin was subjected to theTFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysisof the isolated residue confirmed formation of the selectivelyhydrogenated cyclic dodecapeptide 133. Mass spectrum (ESI⁺, MeCN/H₂O):m/z 792.5 [½(M+2H)]⁺, %(C₇₇H₁₀₈N₂₀O₁₇) requires 792.4; m/z 801.4[½(M+H₂O+2H)]⁺, ½(C₇₇H₁₁₀N₂₀O₁₈) requires 801.4.

7.18.4 Olefin Activation: [2,8]-Saturated Dicarba-[3,12]-Act^(†)Conotoxin ImI:Fmoc-Gly-c[Hag-Act-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Act-NH₂

^(†) Act=Activated sidechain, i.e, crotylglycine (Crt) or allylglycine(Hag)

The resin-bound peptide 133a was subjected to the general crossmetathesis procedure with cis-2-butene (Section 7.5.4) under thefollowing conditions:

Method A:

Resin-peptide 133a (76.0 mg, 55.5 μmol), DCM (5 mL), 2^(nd) generationGrubbs' catalyst (24.0 mg, 28.3 μmol, 50 mol %), cis-2-butene (15 psi),benzoquinone (6.2 mg, 57.4 mol), 50° C., 38 h. At the end of thereaction period, a small aliquot of peptidyl-resin was subjected to theTFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysisof the isolated residue indicated the presence of the desiredbutenolysis product 135. No starting material was evident, however, lowintensity doubly charged higher homologue species separated by m/z+7units were observed.

Mass spectrum (ESI⁺, MeCN/H₂O): m/z 778.4 [½(M+2H)]⁺ _(product),½(C₇₅H₁₀₄N₂₀O₁₇) requires 778.4. Low intensity higher homologue speciesat m/z 785.4, 793.5, 800.4; Very low intensity peaks at m/z 807.8,814.3, 821.3, 828.1, 835.7, 842.6, 849.8, 856.1, 863.6.

Method B:

Resin-peptide 133a (20.0 mg, 10.4 μmol), DCM (3 mL), LiCl/DMF (0.3 mL),2^(nd) generation Grubbs' catalyst (1.0 mg, 1.2 μmol, 10 mol %),cis-2-butene (20 psi), 50° C., 62 h. At the end of the reaction period,a small aliquot of peptidyl-resin was subjected to the TFA-mediatedcleavage procedure (Section 7.3.3). Mass spectral analysis of theisolated residue indicated the presence of the desired product 135, apartially metathesised peptide (mono-crotylglycine containing peptide)136 and the starting peptide 133. Mass spectrum (ESI⁺, MeCN/H₂O): m/z778.4 [½(M+2H)]⁺ _(product), ½(C₇₅H₁₀₄N₂₀O₁₇) requires 778.4. m/z 785.5[½(M+2H)]⁺ ₁₃₆, ½(C₇₆H₁₀₆N₂₀O₁₇); m/z 792.4 [½(M+2H)]⁺ ₁₃₃,½(C₇₇H₁₀₈N₂₀O₁₇); m/z 801.4 [½(M+H₂O+2H)]⁺ ₁₃₃, ½(C₇₇H₁₁₀N₂₀O₁₈).

Method C:

Resin-peptide 133a (35.0 mg, 18 μmol), DCM (5 mL), LiCl/DMF (0.5 mL),2^(nd) generation Grubbs' catalyst (6.2 mg, 7.3 μmol, 40 mol %),cis-2-butene (20 psi), 50° C., 24 h. At the end of the reaction period,a small aliquot of peptidyl-resin was subjected to the TFA-mediatedcleavage procedure (Section 7.3.3). Mass spectral analysis of theisolated residue indicated the presence of the desired product 135, apartially metathesised peptide (mono-crotylglycine containing peptide)136 and the starting peptide 133. Mass spectral data were consistentwith those reported above (Method B).

An analogous reaction was performed for 62 h under the followingconditions: Resin-peptide 133a (30.2 mg, 15.7 μmol), DCM (5 mL),LiCl/DMF (0.5 mL), 2^(nd) generation Grubbs' catalyst (6.2 mg, 7.3 μmol,40 mol %), cis-2-butene (20 psi), 50° C., 62 h. At the end of thereaction period, a small aliquot of peptidyl-resin was subjected to theTFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysisof the isolated residue indicated the presence of the desired product135, a partially metathesised peptide 136 and the starting peptide 133.Mass spectral data were consistent with those previously reported(Method B).

The resin-bound peptide 133a was subjected to the general crossmetathesis procedure with ethylene (Section 7.5.4) under the followingconditions:

Method D:

Resin-peptide 133a (42.0 mg, 21.8 mop, DCM (5 mL), LiCl/DMF (0.5 mL),2^(nd) generation Grubbs' catalyst (7.5 mg, 8.8 μmol, 40 mol %),ethylene (60 psi), 50° C., 62 h. At the end of the reaction period, asmall aliquot of peptidyl-resin was subjected to the TFA-mediatedcleavage procedure (Section 7.3.3). Mass spectral analysis of theisolated residue indicated the presence of the starting peptide 133 anda partially metathesised peptide (mono-allylglycine containing peptide)137. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 778.4 [½(M+2H)]⁺ ₁₃₇,½(C₇₅H₁₀₄N₂₀O₁₇); m/z 792.4 [½(M+2H)]⁺ ₁₃₃.

An analogous reaction was performed in the absence of the chaotropicsalt (LiCl) under the following conditions: Resin-peptide 133a (68.0 mg,35.4 μmol), DCM (5 mL), 2^(nd) generation Grubbs' catalyst (12.0 mg,14.1 μmol, 40 mol %), ethylene (60 psi), 50° C., 62 h. At the end of thereaction period, a small aliquot of peptidyl-resin was subjected to theTFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysisof the isolated residue indicated the presence of the starting peptide133 and a partially metathesised peptide (mono-allylglycine containingpeptide) 137. Mass spectral data were consistent with those reportedabove.

7.18.5 [2,8]-Saturated Dicarba-[3,12]-Dicarba Conotoxin ImI:Fmoc-Gly-c[Hag-c(Crt-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Crt)-NH₂ 140

The Rink amide-bound peptide 135a was subjected to the generalmicrowave-accelerated RCM procedure (Section 7.5.3) under the followingconditions: Resin-peptide 135a (20.0 mg, 14.0 μmol), DCM (5 mL),LiCl/DMF (0.4 M, 0.5 mL), 2^(nd) generation Grubbs' catalyst (2.4 mg,2.8 μmol, 20 mol %), 100° C., 1 h. At the end of the reaction period, asmall aliquot of peptidyl-resin was subjected to the TFA-mediatedcleavage procedure (Section 7.3.3). LC-MS analysis of the isolatedresidue supported formation of the bicyclic peptide 140. LC-MS (Luna C8RP-column, 10-60% MeOH, 0.1% formic acid): t_(R)=9.18 min, m/z 750.4[½(M+2H)]⁺, ½(C₇₁H₉₄N₂₀O₁₇) requires 750.4.

7.18.6 Attempted Synthesis of [2,8]-[3,12]-Saturated Bis-DicarbaConotoxin ImI: NH₂-c[Hag-c(Crt-Ser-Asp-Pro-Arg-Hag]-Ala-Trp-Arg-Crt)-NH₂126

The resin-bound peptide 140a was subjected to the general Wilkinson'shydrogenation procedure (Section 7.4.4) under the following conditions:Resin-peptide 140a (12.2 mg, 8.5 μmol), DCM:MeOH (9:1, 6.5 mL),Wilkinson's catalyst, 80 psi H₂, 22° C., 24 h. At the end of thereaction period, a small aliquot of peptidyl-resin was Fmoc-deprotectedwith 20% piperidine/DMF (1×1 min, 2×20 min). The resin was then washedwith DMF (5×1 min), DCM (3×1 min), MeOH (3×1 min) and dried on the SPPSmanifold for 1 h. The Fmoc-deprotected peptidyl-resin was then subjectedto the TFA-mediated cleavage procedure (Section 7.3.3). Mass spectraland LC-MS data of the isolated residue were inconclusive. The massspectrum and LC-traces did not display peaks due to the fullydeprotected starting peptide 125 and the target saturated bicycle 126.Lack of material and time constraints did not allow us to investigatethis chemistry further.

7.19 [2,8]-[3,12]-Dicarba Conotoxin Transformations 7.19.1 Linear[2,8]-Pre-[3,12]-Hag Conotoxin ImI:Fmoc-Gly-Pre-Hag-Ser-Asp-Pro-Arg-Pre-Ala-Trp-Arg-Hag-NH₂ 128

The procedure described in Section 7.3.2.2 was used for the attachmentof the first amino acid, Fmoc-L-Hag-OH 96, to Rink amide resin.Quantities of the resin and coupling reagents HATU and NMM are presentedin Table 7.16. The first coupling reaction was shaken for 12 h.

TABLE 7.16 Quantities of Reagents used in the Synthesis of Peptide 128Reagent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 705 mg0.37 Fmoc-L-Hag-OH 371 mg 1.10 HATU 280 mg 0.74 NMM 245 μl 2.22

The procedure outlined in Section 7.3.2.2 was also utilised forsubsequent coupling reactions in the synthesis of the dodecapeptide 128.The quantities of successive amino acids and their reaction durationsare detailed in Table 7.17.

TABLE 7.17 Quantities of Amino Acids used in the Synthesis of Peptide128 Amino Acid Mass (mg) Mole (mmol) Reaction Time (h)*Fmoc-L-Arg(Pbf)-OH 715 1.10 2.5 Fmoc-L-Trp(Boc)—OH 580 1.10 2.5Fmoc-L-Ala-OH 343 1.10 2.5 Fmoc-L-Pre-OH 402 1.10 12 Fmoc-L-Arg(Pbf)-OH715 1.10 2.5 Fmoc-L-Pro-OH 371 1.10 2.5 Fmoc-L-Asp(^(t)Bu)—OH 453 1.102.5 Fmoc-L-Ser(^(t)Bu)—OH 422 1.10 12 Fmoc-L-Hag-OH 371 1.10 2.5Fmoc-L-Pre-OH 402 1.10 2.5 Fmoc-L-Gly-OH 328 1.10 2 (12) *Note: Reactiontimes have not been optimised.

After the final amino acid coupling, a small aliquot of peptidyl-resinwas subjected to the TFA-mediated cleavage procedure (Section 7.3.3).Mass spectra analysis of the isolated residue confirmed formation of thedodecapeptide 128. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 805.6 [½(M+2H)]⁺,½(C₇₉H₁₁₀N₂₀O₁₇) requires 805.4; m/z 816.6 [½(M+Na+H)]⁺,½(C₇₉H₁₁₁N₂₀NaO₁₈) requires 816.4.

7.19.2 [2,8]-Pre-[3,12]-Dicarba ConotoxinFmoc-Gly-Pre-c[Hag-Ser-Asp-Pro-Arg-Pre-Ala-Trp-Arg-Hag]-NH₂ 132

Method A:

The resin-bound peptide 128a was subjected to the conventional RCMprocedure (Section 7.5.2) under the following conditions: Resin-peptide128a (19.7 mg, 10.2 μmol), DCM (3 mL), LiCl/DMF (0.4 M, 0.3 mL), 2^(nd)generation Grubbs' catalyst (3.5 mg, 4.1 μmol, 40 mol %), 50° C., 40 h.At the end of the reaction period, a small aliquot of peptidyl-resin wassubjected to the TFA-mediated cleavage procedure (Section 7.3.3). Massspectral analysis of the isolated residue indicated recovery of thelinear peptide 132. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 805.6 [½(M+2H)]⁺_(linear), ½(C₇₉H₁₁₀N₂₀O₁₇).

Method B:

The resin-bound peptide 128a was subjected to the generalmicrowave-accelerated RCM procedure (Section 7.5.3) under the followingconditions: Resin-peptide 128a (550 mg, 0.29 mmol), DCM (5 mL), LiCl/DMF(0.4 M, 0.5 mL), generation Grubbs' catalyst (49.0 mg, 57.7 μmol, 20 mol%), 100° C., 1 h, 100% conversion into 132. At the end of the reactionperiod, a small aliquot of peptidyl-resin was subjected to theTFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysisof the isolated residue confirmed formation of the cyclic peptide 132.Mass spectrum (ESI⁺, MeCN/H₂O): m/z 791.4 [½(M+2H)]⁺, ½(C₇₇H₁₀₆N₂₀O₁₇)requires 791.4.

7.19.3 [3,12]-Pre-[2,8]-Saturated Dicarba Conotoxin:Fmoc-Gly-Pre-c[Hag-Ser-Asp-Pro-Arg-Pre-Ala-Trp-Arg-Hag]-NH₂ 134

The resin-bound peptide 132a was subjected to the general Wilkinson'shydrogenation procedure (Section 7.4.4) under the following conditions:Resin-peptide 132a (365 mg, 0.19 mmol), DCM:MeOH (9:1, 5 mL),Wilkinson's catalyst, 80 psi H₂, 22° C., 19 h. At the end of thereaction period, a small aliquot of peptidyl-resin was subjected to theTFA-mediated cleavage procedure (Section 7.3.3). Mass spectral analysisof the isolated residue confirmed formation of the selectivelyhydrogenated cyclic peptide 134. Mass spectrum (ESI⁺, MeCN/H₂O): m/z792.5 [½(M+2H)]⁺, ½(C₇₇H₁₀₈N₂₀O₁₇) requires 792.4; m/z 801.4[½(M+H₂O+2H)]⁺, ½(C₇₇H₁₁₀N₂₀O₁₈) requires 801.4.

7.19.4 Attempted Synthesis ofFmoc-Gly-Crt-c[Hag-Ser-Asp-Pro-Arg-Crt-Ala-Trp-Arg-Hag]-NH₂ 138

The resin-bound peptide 134a was subjected to the conventional crossmetathesis procedure with cis-2-butene (Section 7.5.4) under thefollowing conditions:

Method A:

Resin-peptide 134a (78.5 mg, 41 μmol), DCM (5 mL), 2^(nd) generationGrubbs' catalyst (13.9 mg, 16 μmol, 40 mol %), cis-2-butene (15 psi),50° C., 62 h. At the end of the reaction period, a small aliquot ofpeptidyl-resin was subjected to the TFA-mediated cleavage procedure(Section 7.3.3). Mass spectral analysis of the isolated residueindicated the presence of a mixture of peptides: the starting peptide134, the desired product 138 and a partially metathesised peptide(mono-butenolysis product) 139. Mass spectrum (ESI⁺, MeCN/H₂O): m/z778.5 [½(M+2H)]⁺ _(product), ½(C₇₅H₁₀₄N₂₀O₁₇) requires 778.4; m/z 785.5[½(M+2H)]⁺ ₁₃₉, ½(C₇₆H₁₀₆N₂₀O₁₇); m/z 792.5 [½(M+2H)]⁺ ₁₃₄,½(C₇₇H₁₀₈N₂₀O₁₇).

An analogous reaction in the presence of a chaotropic salt (LiCl) wasperformed under the following conditions: Resin-peptide 134a (60.1 mg,31 μmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2^(nd) generationGrubbs' catalyst (10.6 mg, 12 μmol, 40 mol %), cis-2-butene (15 psi),50° C., 62 h. At the end of the reaction period, a small aliquot ofpeptidyl-resin was subjected to the TFA-mediated cleavage procedure(Section 7.3.3). Mass spectral analysis of the isolated residueindicated the presence of a mixture of peptides: the starting peptide134a, the desired product 138 and a partially metathesised peptide 139.Mass spectral data were consistent with those reported above.

7.19.5 Linear [2,8]-Pre-[3,12]-Hag Conotoxin ImI (Ala9→Pro9Replacement): Fmoc-Gly-Pre-Hag-Ser-Asp-Pro-Arg-Pre-Ala-Trp-Arg-Hag-NH₂130

The procedure described in Section 7.3.2.2 was used for the attachmentof the first amino acid, Fmoc-L-Hag-OH 96, to Rink amide resin.Quantities of the resin and coupling reagents HATU and NMM are presentedin Table 7.18. The first coupling reaction was shaken for 5 h.

TABLE 7.18 Quantities of Reagents used in the Synthesis of Peptide 130Reagent Mass (mg) or Volume (μl) Mole (mmol) Rink Amide Resin 400 mg0.29 Fmoc-L-Hag-OH 295 mg 0.88 HATU 223 mg 0.59 NMM 193 μl 1.75

The procedure outlined in Section 7.3.2.2 was also utilised forsubsequent coupling reactions in the synthesis of the dodecapeptide 130.The quantities of successive amino acids and their reaction durationsare detailed in Table 7.19.

TABLE 7.19 Quantities of Amino Acids used in the Synthesis of Peptide130 Amino Acid Mass (mg) Mole (mmol) Reaction Time (h)*Fmoc-L-Arg(Pbf)-OH 570 0.88 12  Fmoc-L-Trp(Boc)—OH 462 0.88 2 (1)Fmoc-L-Pro-OH 296 0.88  1 (12) Fmoc-L-Pre-OH 328 0.90 4Fmoc-L-Arg(Pbf)-OH 570 0.88  2 (12) Fmoc-L-Pro-OH 296 0.88 2 (2)Fmoc-L-Asp(^(t)Bu)—OH 361 0.88 2 (2) Fmoc-L-Ser(^(t)Bu)—OH 336 0.88  2(12) Fmoc-L-Hag-OH 296 0.88 4 Fmoc-L-Pre-OH 330 0.90 1 (2) Fmoc-L-Gly-OH262 0.88 2 (1) *Note: Reaction times have not been optimised.

After the final amino acid coupling, a small aliquot of peptidyl-resinwas subjected to the TFA-mediated cleavage procedure (Section 7.3.3).Mass spectral analysis of the isolated residue confirmed formation ofthe dodecapeptide 130. Mass spectrum (ESI⁺, MeCN/H₂O): m/z 818.6[½(M+2H)]⁺, ½(C₈₁H₁₁₂N₂₀O₁₇) requires 818.4; m/z 827.6 [½(M+H₂O+2H)]⁺,½(C₈₁H₁₁₄N₂₀O₁₈) requires 827.4.

7.19.6 [2,8]-Pre-[3,12]-Dicarba Conotoxin ImI (Ala9→Pro9 replacement):Fmoc-Gly-Pre-c[Hag-Ser-Asp-Pro-Arg-Pre-Ala-Trp-Arg-Hag]-NH₂ 131

The resin-bound peptide 130a was subjected to the conventional RCMprocedure (Section 7.5.2) under the following conditions: Resin-peptide130a (97.0 mg, 70.8 μmol), DCM (5 mL), LiCl/DMF (0.4 M, 0.5 mL), 2^(nd)generation Grubbs' catalyst (24.1 mg, 28.4 μmol, 40 mol %), 50° C., 40h. At the end of the reaction period, a small aliquot of peptidyl-resinwas subjected to the TFA-mediated cleavage procedure (Section 7.3.3).Mass spectral analysis of the isolated residue confirmed the presence ofboth cyclic 131 and linear 130 peptides. Mass spectrum (ESI⁺, MeCN/H₂O):m/z 804.5 [½(M+²H)]⁺ _(cyclic), ½(C₇₉H₁₀₈N₂₀O₁₇) requires 804.4; m/z813.8 [½(M+H₂O+2H)]⁺ _(cyclic), ½(C₇₉H₁₁₀N₂₀O₁₈) requires 813.4; m/z818.7 [½(M+2H)]⁺ _(linear), ½(C₈₁H₁₁₂N₂₀O₁₇); m/z 827.3 [½(M+H₂O+2H)]⁺_(linear), ½(C₈₁H₁₁₄N₂₀O₁₈).

7.20 Activation Studies 7.20.1 6-Acetoxy-2-benzamido-4-hexenoic acidmethyl ester 141

Standard solution phase metathesis conditions (see section 7.5) wereemployed to synthesise 6-acetoxy-2-benzoylamino-4-hexenoic acid methylester 141 from the cross metathesis of the corresponding prenylderivative 87 and 1,4-diacetoxy-cis-2-butene. The desired product wasobtained as a dark brown oil following by column chromatography (SiO₂;EtOAc:Hexane, 1:1).

N-Bzl-O-Me-prenylglycine (170 mg, 0.65 mmol), dichloromethane (10 mL),second generation Grubbs' catalyst (16.5 mg, 5 mol %, 0.03 mmol),1,4-diacetoxy-cis-2-butene (671.5 mg, 3.9 mmol), 50° C., 20 h; 112.5 mg,57%.

GC: t_(R) (E/Z)=12.96, 13.06 min (GC column 30QC5/BPX5, 150° C. for 1min, 10° C. min⁻¹ to 280° C. for 6 min.)

IR (film): 3333s; 3056w; 3015w; 2944s; 1739s; 1662m; 1641s; 1605m;1574m; 1533s; 1487m; 1436m; 1364m; 1236s; 1154w; 1072w; 1026m; 969m;801w; 718m; 692w cm⁻¹.

¹H NMR (400 MHz, CDCl₃): δ 2.00, s, 3H, CH₃; 2.67, m, 2H, H3; 3.77, s,3H, OCH₃; 4.49, d, J 4.7 Hz, 2H, H6; 4.89, q, J 5.8 Hz, 1H, H2; 5.68, t,J 5.2 Hz, 2H, H4, 5; 6.75, d, J 7.4 Hz, 2H, H4, 5; 7.42, t, J 7.2 Hz,2H, H4′, 6′; 7.50, t, J 6.4 Hz, 1H, H5′; 7.78, d, J 7.1 Hz, 2H, H3′, 7′.

¹³C NMR (125 MHz, CDCl₃): δ 20.9, CH₃; 35.2, C3; 52.1, OCH₃; 52.7, C2;64.5, C6; 127.2, C3′, 7; 128.7, C4′, 6′; 128.9, C5; 129.1, C4; 131.9,C5′; 133.9, CT; 167.1, C1′; 170.8, C1″; 172.3, C1.

Mass Spectrum (ES CH₃CN): m/z 328.1 (M+Na⁺) C₁₆H₁₉NO₅Na.

HRMS (EI, CH₃OH). found m/z 305.1263, C₁₆H₁₉NO₅ requires 305.1263.

7.20.2 2,7-Bis-benzamido-oct-4-enedioic acid dimethyl ester 69

2,7-Bis-benzoylamino-4-octenedioic acid dimethyl ester was synthesisedusing standard solution phase metathesis conditions (refer to section7.5). Due to the equilibrium generated in the reaction, a mixture of thehomodimer 69 and the starting material 141 was obtained.

6-Acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 141 (53.5 mg, 0.18mmol), dichloromethane (10 mL), second generation Grubbs' catalyst (5mol %, 7.4 mg, 8.8 μmol), 50° C., 18 h.

GC: t_(R) (1,4-diacetoxy-cis-2-butene)=3.28; (product E/Z)=13.18, 13.31min (GC column 30QC5/BPX5, 150° C. for 1 min, 10° C. min⁻¹ to 280° C.for 6 min.). The mass spectrum was consistent with that previouslydescribed for this compound.

7.20.3 2-Acetylamino-7-benzoylamino-4-octenedioic acid dimethyl ester142

2-Acetylamino-7-benzoylamino-4-octenedioic acid dimethyl ester 142 wassynthesised using standard solution-phase metathesis conditions (referto section 7.5) from 6-acetoxy-2-benzoylamino-4-hexenoic acid methylester 141 and methyl-2-acetylamino-4-pentenoate 121a. The desiredcompound was obtained as a brown oil, and purified via columnchromatography (SiO₂; EtOAc:Hexane; 2:1).

6-Acetoxy-2-benzoylamino-4-hexenoic acid methyl ester 141 (50 mg, 0.16mmol), dichloromethane (10 mL), second generation Grubbs' catalyst (7mg, 5 mol %, 8 μmol), methyl-2-acetylamino-4-pentenoate 142 (168 mg,0.98 mmol), 50° C., 18 h, 48.6 mg, 81%.

GC: t_(R) (E/Z)=14.30, 14.50 min (GC column 30QC5/BPX5, 150° C. for 1min, 10° C. min⁻¹ to 280° C. for 6 min.)

¹H NMR (500 MHz, CDCl₃, mixture of isomers (1:1.2)): δ 1.95, s (majorisomer) and 1.97, s (minor isomer), 3H, CH₃; 2.42-2.70, m, 4H, H3, 6;3.62, s (minor isomer), 3.64, s (major isomer), 3.78, s (minor isomer)and 3.79, s (major isomer), 6H, 2×OCH₃; 4.63-4.66, m, 1H, H2; 4.85-4.91,m, 1H, H7; 5.35-5.49, m, 2H, H4, 5; 6.20, d, J 7.7 Hz (major isomer) and6.34, d, J 7.5 Hz, 1H, NH (minor isomer); 6.87, t, J 7.55 Hz, 1H, NH;7.44, t, J 7.1 Hz, 2H, H4′, 6′; 7.50, t, J 6.9 Hz, 1H, H5′; 7.84, t, J7.9 Hz, 2H, H3′, 7′.

¹³C NMR (75 MHz, CDCl₃): δ 22.8, CH₃; 34.8, 35.1, 35.4 and 35.7, C3, 6;51.5 and 51.6, C2; 52.4, 52.5, 52.5, 52.6 and 52.7, C7, 2×OCH₃; 127.2and 127.2, C3′, 7′; 128.6 and 128.6, C4′, 6′; 128.9 and 129.0, C4, 5;131.9 and 131.9, C5′; 133.7, C2; 167.1, COPh; 170.0 and 170.1, COMe;172.2, 172.3, 172.3 and 172.4, 2×COOMe.

Mass Spectrum (ESI⁺, CH₃OH): m/z 399.2 (M+Na⁺) C₁₉H₂₄N₂O₆Na.

7.20.4 Synthesis of Fmoc-Pre-Phe-OH on Wang Resin

The dipeptide, Fmoc-Pre-Phe-OH, was synthesised on pre-functionalisedFmoc-Phe-Wang resin (250 mg, 0.13 mmol) according to standard SPPStechniques (see section 7.3.2). Fmoc-prenylglycine (138 mg, 0.38 mmol)was coupled using HATU (144.5 mg, 0.38 mmol) and NMM (83.6 mL, 0.76mmol). An aliquot of resin was subjected to cleavage condtions (seesection 7.3.3) to assess reaction success.

Mass Spectrum (ESI+, CH₃OH): m/z 513.2 (M+H⁺), +), C₃₁H₃₃N₂O₅; 535.1(M+Na+), C₃₁H₃₂N₂O₅Na.

7.20.5 Activation of Resin-Bound Prenylglycine

The resin-tethered dipeptide was subjected to microwave-assisted crossmetathesis conditions (see section 7.3.2) with1,4-diacetoxy-cis-2-butene. An aliquot of resin was subjected tocleavage condtions (see section 7.3.3) to assess reaction success.

Resin-tethered dipeptide (180 mg, 0.09 mmol), second generation Grubbs'catalyst (15.3 mg, 20 mol %, 0.018 mmol), 1,4-diacetoxy-cis-2-butene (97mg, 0.56 mmol), dichloromethane (10 mL), 100° C., 1 h, 100% conversion.

Mass Spectrum (ESI+, CH₃OH): m/z 557.2 (M+H⁺), +), C₃₂H₃₃N₂O₇; 579.2(M+Na+), C₃₂H₃₂N₂O₇Na.

7.21 Dicarba AOD Studies 7.21.1 Manual Synthesis of Linear AOD9604 146

The manual peptide synthesis procedure described in Section 7.3.2 wasused for the synthesis of AOD9604 on Wang-Phe-Fmoc resin. Quantities ofthe resin and coupling reagents HATU and NMM are tabulated below. Thequantities of successive amino acids are summarized below:

Mole Compound Quantity MW or Loading (mmol) Equivalents Wang-Phe-Fmoc500 mg 0.52 mmol/g 0.25 1 Resin HATU 198 mg 380.23 0.52 2 NMM 172 μL101.15 1.56 6 Quantity Mole Reaction Compound (mg) MW (mmol)/Eq. Time(hr) Fmoc-Gly-OH 232 297.14 0.78/3 2 Fmoc-Hag-OH 263 337.37 0.78/3 16Fmoc-Gly-Ser(ψPro)- 331 424.5 0.78/3 2 OH Fmoc-Glu-OH 332 425.5 0.78/3 2Fmoc-Val-OH 265 339.22 0.78/3 2 Fmoc-Ser-OH 299 383.4 0.78/3 2Fmoc-Arg-OH 534 684.4 0.78/3 16 Fmoc-Hag-OH 263 337.37 0.78/3 2Fmoc-Gln-OH 476 610.7 0.78/3 2 Fmoc-Val-OH 265 339.22 0.78/3 2Fmoc-Ile-OH 276 353.24 0.78/3 2 Fmoc-Arg-OH 534 684.8 0.78/3 16Fmoc-Leu-OH 276 353.24 0.78/3 2 Fmoc-Tyr-OH 358 459.5 0.78/3 2

Following the final amino acid coupling, a small aliquot of the resinbound peptide was cleaved as described in Section 7.3.3 for mass specanalysis. Mass spectrum (ESI⁺, MeOH/H₂O): m/z 676.5 (M+3H/3), m/z 1014.6(M+2H/2).

7.21.2 Automated Synthesis of Linear AOD9604 146

The procedure described in Section 7.5.3 was used for the synthesis ofAOD9604 on Wang-Phe-Fmoc resin. Quantities of the resin, couplingreagents and amino acids are tabulated below:

Mole Quantity (mmol)/ Compound (mL/g) Volume Conc (M) Cycle NameWang-Phe- 0.481 g 5 mL DMF 0.25 mmol — Fmoc DIPEA  7.7 mL 22 mL NMP 2M —HBTU 6.827 g 36 mL DMF 0.45M — HOBt 2.432 g Fmoc-Arg- 1.427 g 11 mL DMF0.2M B0.25-Double OH (ext.) Fmoc-Hag- 1.289 g 11 mL DMF 0.2MB0.25-Single (ext.) OH Fmoc-Gln- 0.737 g 6 mL DMF 0.2M B0.25-Single(ext.) OH Fmoc-Glu- 0.425 g 5 mL DMF 0.2M B0.25-Single (ext.) OHFmoc-Gly- 0.357 g 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Ile- 0.424 g6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Leu- 0.353 g 5 mL DMF 0.2MB0.25-Single (ext.) OH Fmoc-ψPro- 0.562 g 6 mL DMF 0.2M B0.25-Single(ext.) OH Fmoc-Ser- 0.460 g 6 mL DMF 0.2M B0.25-Single (ext.) OHFmoc-Tyr- 0.551 g 6 mL DMF 0.2M B0.25-Single (ext.) OH Fmoc-Val- 0.747 g11 mL DMF 0.2M B0.25-Single (ext.) OH

Resin washings and deprotection cycles were performed as described inSection 7.5.3. The amino acid, activator and activator base solutionswere added to the resin, followed by the “B.01 Extended Coupling” cycle.The peptidyl-resin was exposed to a temperature of 75° C. with no power(0 watts) for 2 min, then at a temperature of 75° C., power at 25 wattsfor 10 min. The peptidyl-resin was then washed with DMF (3×10 mL). Mostamino acids are programmed with the “B0.25-Single (Extended)” couplingcycle, which concludes at this point. However arginine requires and“B0.25-Double (Extended)” coupling cycle as several AOD deletionproducts have been produced in the past. This involves two “B0.1Extended Coupling” cycle programs.

Following the final amino acid coupling, a small aliquot of the resinbound peptide was cleaved as described in Section 7.3.3 for mass specanalysis. Mass spectrum (ESI⁺, MeOH/H₂O): m/z 689.1 (M+3H/3), m/z 1014.2(M+2H/2).

7.21.3 Ring Closing Metathesis of Linear AOD9604 146

The resin bound peptide 146 was subjected to microwave RCM procedureoutlined in section 7.5.3. Peptidyl-resin (0.9088 mg, 0.475 mmol) and2^(nd) generation Grubb's catalyst (80 mg, 0.095 mmol) was weighted intoa glass vial loaded with stirrer bar. In a drybox, DCM (5 mL) andLiCl/DMF (0.2 mL) were added and the vial was sealed. The reactionvessel was placed in the microwave for 1 hr at 100 C. A small aliquot ofthe resin bound peptide was subjected to TFA cleavage and analysed bymass spec to show the target unsaturated AOD 147. Mass spectrum (ESI⁺,MeOH/H₂O): m/z 1000.3 (SM+2H/2), m/z 1014.2 (M+2H/2). The crude peptidewas purified by reverse phase HPLC.

7.21.4 Hydrogenation of Unsaturated Cyclic AOD 147

A 150 mL glass hydrogenation vessel with plastic shield was loaded withthe peptidyl-resin (0.9168 mg, 0.476 mmol) and stirrer bar. In an inertatmosphere, Wilkinson's catalyst (22 mg, 0.024 mmol) and 10 mL solvent(DCM:MeOH, 9:1) was added. The vessel was sealed with a rubber 0 ringand fitted with a pressure regulator. The vessel was purged with argonthen hydrogen to a pressure of 90 psi and reacted at r.t. for 4 days.The reaction was terminated upon exposure to oxygen and the resin waswashed with DCM (5 mL, 3×1 min), DMF (5 mL, 3×1 min) then MeOH (5 mL,3×1 min) and dried in vacuo for 30 min prior to cleavage and mass specanalysis. Mass spec analysis showed conversion to the saturated cyclicpeptide 148 (ESI⁺, MeOH/H₂O): m/z 1000.9 (M+2H/2), m/z 1015.6 (SM+2H/2).The crude peptide was purified by reverse phase HPLC.

8.0 BIOLOGICAL TESTING

Bovine adrenal chromaffin cells can be used to test for the activity ofα-CTX ImI at neuronal-type nicotinic receptors. These cells are of twotypes, adrenaline- and noradrenaline-containing, and possess theneuronal-type nicotinic receptor subtypes α3β4 and α7. When stimulatedwith nicotine, these cells release adrenaline and noradrenaline whichcan be measured. Native α-CTX ImI peptides inhibit thenicotine-stimulated release of these neurotransmitters by interactingwith the α3β4-receptor subtype.

Dicarba-conotoxins 118 and 119 were assayed in quadruplicate inmultiwells (6×4) containing monolayer cultures of bovine adrenalchromaffin cells as described by Broxton et al. (Loughnan, M., Bond, T.,Atkins, A., Cuevas, J., Adams, D. J., Broxton, N.M., Livett, B. G.,Down, J. G., Jones, A., Alewood, P. F., Lewis, R. J. J. Biol. Chem.,1998, 273 (25), 15667-15674.)^(X) The response of the cells to thesepeptides was tested at two peptide concentrations 1 uM and 5 uM. Thecells were stimulated with nicotine (4 uM) for 5 min at room temp (23C)and the amount of catecholamine release (noradrenaline and adrenaline)was measured over a 5 period and expressed as a % of the initialcellular content of these amines. This was performed in the presence andabsence (control) of dicarba-conotoxin peptides. The chromaffin cellsalso leak small amounts of catecholamine over the measurement period, soa ‘basal release’ (no nicotine added) measurement is also recorded.

FIG. 4 shows catecholamine release from dicarba-conotoxins 118 and 119.Basal release was measured at 0.72% (and is subtracted from othermeasurements). Nicotine stimulation alone released (6.85-0.72)=6.13% ofthe noradrenaline in the cells, but only (4.15-0.72)=3.43% in thepresence of 5 uM of [2,8]-cystino-[3,12]-dicarba conotoxin 119. Thisrepresents 55.97% of the release produced by nicotine alone, or a 44%inhibition of release (see Table). The % inhibition of release ofadrenaline (41%) was similar to that for noradrenaline (44%). Thisinhibition was found to be concentration related: A 1 uM sample ofdicarba-conotoxin 119 produced only a 21.6% inhibition of noradrenalinerelease and a 16.5% inhibition of release of adrenaline.

Data for [2,8]-dicarba-[3,12]-cystino conotoxin 118 is also shown inFIG. 4 and Table 8.1. This data shows that these dicarba-analogues arebiologically active and possess activity profiles analogous to thenative conotoxin sequences.

TABLE 8.1 Catecholamine release for dicarba-conotoxins 118 and 119 1st2nd 3rd 4th mean SEM n % control % inhibition Noradrenaline Release BASE0.68 0.61 0.66 0.93 0.72 0.07 4 NICOTINE (4 uM) 6.62 6.89 6.99 6.89 6.850.08 4 100.000 119 (1 uM) 4.89 5.75 5.73 5.73 5.52 0.21 4 78.400 21.600119 (5 uM) 3.22 4.58 3.99 4.81 4.15 0.35 4 55.971 44.029 118 (1 uM) 4.526.29 7.00 7.34 6.29 0.63 4 90.859 9.141 118 (5 uM) 4.19 5.86 4.32 5.514.97 0.42 4 69.384 30.616 Adrenaline Release BASE 0.38 0.28 0.31 0.340.33 0.02 4 NICOTINE (4 uM) 4.31 4.52 4.52 4.48 4.46 0.05 4 100.000 119(1 uM) 3.31 3.67 4.14 3.98 3.77 0.18 4 83.420 16.580 119 (5 uM) 2.322.95 2.76 3.02 2.76 0.16 4 58.888 41.112 118 (1 uM) 2.94 4.40 4.46 4.093.97 0.35 4 88.260 11.740 118 (5 uM) 2.55 3.63 2.62 3.31 3.03 0.26 465.306 34.694

9.0 STABILITY 9.1 Thiol Stability

Peptides samples (0.25 mM) were dissolved in a solution containingeither 0.25 mM reduced glutathione, 12.3 μM reduced thioredoxin(Promega, Madison, Wis.) or 0.5 mM human serum albumin (Sigma, Madison,Wis.) in 100 mM phosphate buffer+1 mM EDTA, pH 7.4 (300 μL) andincubated at 37 C. Thioredoxin was reduced by treating the oxidised formwith 0.9 equivalents of dithiothreitol for 15 minutes immediately priorto use. Aliquots (30 μL) were taken at various time intervals, quenchedwith extraction buffer consisting of 50% aqueous acetonitrile, 100 mMNaCl, and 1% TFA (30 μL) and analysed by reverse-phase-HPLC. The ratioof the degradation product to the tested peptide sample was determinedby measuring the peak height, and compared against the peak heightresults for the HPLC of the corresponding natural or native peptide. Theproduct was considered to have improved stability if the comparativeHPLC test results showed less degradation product after 6 hours ofcontact with one of the agents (reduced glutathione, reduced thioredoxinor human serum albumin).

9.2 Human Blood Plasma Stability

Whole human blood containing 1% EDTA was centrifuged at 14,000 rpm for30 minutes. The supernatant was then transferred to an Eppendorf tubeand centrifuged for an additional 30 min at 14,000 rpm. Peptide sampleswere dissolved in plasma (200 μL) to an initial peptide concentration ofapproximately 0.25 mM. Aliquots (30 μL) were removed at various timeintervals and quenched with extraction buffer (30 μL). The aliquot wasthen vortexed, diluted with additional water (60 μL) and chilled in anice bath for 5 minutes prior to centrifuging at 14,000 rpm for 15minutes. The supernatant was then analysed by RP-HPLC. The stability ofthe peptide sample was assessed by comparing the ratio of the peakheights representing the tested peptide, and the degradation products,against a sample of the corresponding natural or native peptide notcontaining the dicarba bridge or bridges. The product was considered tohave improved stability in human blood plasma if the comparative HPLCtest results showed less degradation product after 6 hours of contact.

It will be understood to persons skilled in the art of the inventionthat many modifications may be made without departing from the spiritand scope of the invention.

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A.1 The Amino Acids One Three letter letter Amino acid code codeStructure Alanine A Ala

Allylglycine* — Hag

Arginine R Arg

Asparagine N Asn

Aspartic acid D Asp

Crotylglycine* — Crt

Cysteine C Cys

5,5-Dimethylproline* — dmP

Glutamic acid E Glu

Glutamine Q Gln

Glycine G Gly

Histidine H His

Isoleucine I Ile

Leucine L Leu

Lysine K Lys

Methionine M Met

Phenylalanine F Phe

Prenylglycine* — Pre

Proline P Pro

Serine S Ser

Threonine T Thr

Tryptophan W Trp

Tyrosine Y Tyr

Valine V Val

*Synthetic amino acids.

The claims defining the invention are as follows:
 1. A method for thesynthesis of an organic compound with a dicarba bridge, comprising:providing a reactable organic compound having a pair of unblockedcomplementary metathesisable groups, or two or more reactable organiccompounds having between them a pair of unblocked complementarymetathesisable groups, and subjecting the reactable organic compound orcompounds to cross-metathesis under microwave radiation conditions toform an organic compound with an unsaturated dicarba bridge.
 2. Themethod of claim 1, further comprising: hydrogenating the unsaturateddicarba bridge.
 3. The method of claim 1 or claim 2, wherein thecomplementary metathesisable groups of the first pair of complementarymetathesisable groups are each independently selected from the groupconsisting of olefins comprising the portion ═CH₂, and monosubstitutedolefins comprising the group ═CHR₅, in which R₅ is alkyl or an alkylsubstituted by a polar functional group.
 4. The method of any one ofclaims 1 to 3, wherein the reactable organic compound, or one of thereactable organic compounds, is attached to a solid support during thecross-metathesis of the complementary metathesisable groups.
 5. Themethod of claim 4, wherein the loading of the reactable organic compoundon the solid support is at least 0.2 mmol/g.
 6. The method of any one ofclaims 4 to 5, wherein each cross-metathesis is performed using across-metathesis catalyst, and each cross-metathesis is performed in asolvent combination of a resin-swelling solvent, with a co-ordinatingsolvent for the catalyst.
 7. The method of claim 6, wherein theco-ordinating solvent is an alcohol.
 8. The method of claim 6 or claim7, wherein the co-ordinating solvent is used in an amount of about 1-30%by volume, with respect to the total solvent combination.
 9. The methodof any on eof claims 1 to 8, wherein the reactable organic compound, orthe two or more reactable organic compounds, between them contain asecond pair of complementary metathesisable groups which are blocked,and can be unblocked by an unblocking reaction specific to the secondpair of complementary metathesisable groups, and the method furthercomprises unblocking the blocked second pair of complementarymetathesisable groups, followed by cross-metathesis of the second pairof cross-methatesisable groups.
 10. The method of claim 9, wherein theblocked second pair of complementary metathesisable groups comprisedialkyl-blocked olefins.
 11. The method of claim 9 or claim 10, whereinthe blocked second pair of complementary metathesisable groups areunblocked by cross-methatiesis with a disposable olefin, which is1,3-butadiene-free.
 12. The method of claim 11, wherein the disposableolefin is a 1,3-butadiene-free olefin or olefin mixture of one or moreof the following:

wherein X and Y are each independently selected from the groupconsisting of H, alkyl and substituted alkyl, wherein the substituent ofthe substituted alkyl is selected from one or more of halo, hydroxy,alkoxy, nitrile, acid and ester.
 13. The method of any one of claims 9to 12, wherein the reactable organic compound comprises a third pair ofcomplementary methathesisable groups, which are blocked and can beunblocked by an unblocking reaction or series of reactions specific tothe third pair, and the method comprises subjecting the third pair ofcomplementary metathesisable groups to unblocking reactions specific tothose pairs, followed by cross metathesis.
 14. The method of claim 13,wherein the blocking group of the third pair of complementarymetathesisable groups comprises an electronic blocking group, and theunblocking reactions comprise conversion of the electronic stericblocking group to a group that is cross-metathesisable.
 15. The methodof claim 14, wherein the electronic blocking group comprises a diblockedconjugated diene, and the unblocking reaction comprises subjecting theblocked third pair of complementary metathesisable groups to asymmetrichydrogenation to regioselectively, stereoselectively andchemoselectively hydrogenate one of the diene double bonds to leave asterically-blocked double bond, followed by cross-metathesis with adisposable olefin to effect removal of the steric blocking groups on theremaining double bond, to yield unblocked complementary metathesisablegroups.
 16. The method of claim 14, wherein the electronic blockinggroup comprises ═CH—CH═CR₃R₄, in which R₃ and R₄ are each alkyl, and theunblocking reaction comprises hydrogenation of this group to═CH—CH₂—CHR₃R₄, followed by cross-metathesis with a disposable olefin toyield the unblocked group ═CHR₅, in which R₅ is alkyl or an alkylsubstituted by a polar functional group.
 17. The method of any one ofclaims 1 to 16, wherein the reactable organic compound is a peptide. 18.The method of claim 17, wherein the organic compound with two or moredicarba bridges prepared by the method is a peptidomimetic.
 19. Themethod of claim 17, wherein the organic compound with two or moredicarba bridges prepared by the method is a pseudopeptide.
 20. Themethod of any one of claims 1 to 19, wherein the reactable organiccompound comprises a peptide comprising a series of amino acidssupported on a solid support, wherein two of the amino acids comprisesidechains with a first pair of complementary metathesisable groups. 21.The method of claim 20, wherein the peptide contains at least oneturn-inducing amino acid between the amino acids that comprise the firstpair of complementary metathesisable groups.
 22. The method of claim 21,wherein the turn-inducing amino acids are selected from one or more ofψserine, ψthreonine and ψcysteine.
 23. The method of claim 22, furthercomprising cleaving the protein from the solid support and convertingany ψserine, ψthreonine and ψcysteine residues present into serine,threonine and cysteine, respectively.
 24. A method for the synthesis ofan organic compound with a dicarba bridge, comprising: synthesising areactable organic compound to contain a pair of unblocked complementarymetathesisable groups, and a turn-inducing group in between the pair ofcomplementary metathesisable groups, and subjecting the reactableorganic compound to cross-metathesis to form a compound with anunsaturated dicarba bridge.
 25. The method of claim 24, furthercomprising hydrogenating the unsaturated dicarba bridge to form anorganic compound with a saturated dicarba bridge.
 26. The method of anyone of claims 24 to 25, wherein the reactable organic compound comprisesa peptide comprising a series of amino acids supported on a solidsupport, wherein two of the amino acids comprise sidechains with a firstpair of complementary metathesisable groups.
 27. The method of claim 26,wherein the peptide contains at least one turn-inducing amino acidbetween the amino acids that comprise the first pair of complementarymetathesisable groups.
 28. The method of claim 27, wherein theturn-inducing amino acids are selected from one or more of ψserine,ψthreonine and ψcysteine.
 29. The method of any one of claims 26 to 28,wherein the complementary metathesisable groups of the first pair ofcomplementary metathesisable groups are each independently selected fromthe group consisting of olefins comprising the portion ═CH₂, andmonosubstituted olefins comprising the group ═CHR₅, in which R₅ is alkylor an alkyl substituted by a polar functional group.
 30. The method ofany one of claims 26 to 29, wherein the loading of the reactable organiccompound on the solid support is at least 0.2 mmol/g.
 31. The method ofany one of claims 26 to 30, wherein each cross-metathesis is performedusing a cross-metathesis catalyst, and each cross-metathesis isperformed in a solvent combination of a resin-swelling solvent, with aco-ordinating solvent for the catalyst.
 32. The method of claim 31,wherein the co-ordinating solvent is an alcohol.
 33. The method of claim31 or claim 32, wherein the co-ordinating solvent is used in an amountof about 1-30% by volume, with respect to the total solvent combination.34. An organic compound or a peptide containing a dicarba bridge whenproduced by the method of any one of claims 1 to
 33. 35. Fmoc-protectedprenyl glycine.
 36. A method for the synthesis of Fmoc-protected prenylglycine, the method comprising cross-metathesis of Fmoc-protected allylglycine with 2-alkyl-2-butylene in the presence of a cross-metathesiscatalyst.